WO2008125599A1 - 2-carboxy thiophene derivatives as anti-viral agents - Google Patents

Abstract

Anti-viral agents of compounds of Formula (I) : wherein A, Rx, Ry, R2 and R3 are as defined in the specification, processes for their preparation and their use in HCV treatment are provided.

Description

2-CARBOXY THIOPHENE DERIVATIVES AS ANTI-VIRAL AGENTS

FIELD OF THE INVENTION

The present invention relates to novel 2-carboxy thiophene derivatives useful as anti-viral agents. Specifically, the present invention involves novel inhibitors of Hepatitis C Virus (HCV) replication.

BACKGROUND OF THE INVENTION

Infection with HCV is a major cause of human liver disease throughout the world. In the US, an estimated 4.5 million Americans are chronically infected with HCV. Although only 30% of acute infections are symptomatic, greater than 85% of infected individuals develop chronic, persistent infection. Treatment costs for HCV infection have been estimated at $5.46 billion for the US in 1997. Worldwide over 200 million people are estimated to be infected chronically. HCV infection is responsible for 40-60% of all chronic liver disease and 30% of all liver transplants. Chronic HCV infection accounts for 30% of all cirrhosis, end-stage liver disease, and liver cancer in the U.S. The CDC estimates that the number of deaths due to

HCV will minimally increase to 38,000/year by the year 2010.

Due to the high degree of variability in the viral surface antigens, existence of multiple viral genotypes, and demonstrated specificity of immunity, the development of a successful vaccine in the near future is unlikely. Alpha-interferon (alone or in combination with ribavirin) has been widely used since its approval for treatment of chronic HCV infection. However, adverse side effects are commonly associated with this treatment: flu-like symptoms, leukopenia, thrombocytopenia, depression from interferon, as well as anemia induced by ribavirin (Lindsay, K. L. (1997) Hepatology 26 (suppl 1 ): 71 S-77S). This therapy remains less effective against infections caused by HCV genotype 1 (which constitutes -75% of all HCV infections in the developed markets) compared to infections caused by the other 5 major HCV genotypes. Unfortunately, only -50-80% of the patients respond to this treatment (measured by a reduction in serum HCV RNA levels and normalization of liver enzymes) and, of responders, 50-70% relapse within 6 months of cessation of treatment. Recently, with the introduction of pegylated interferon (Peg-IFN), both initial and sustained response rates have improved substantially, and combination treatment of Peg-IFN with ribavirin constitutes the gold standard for therapy. However, the side effects associated with combination therapy and the impaired response in patients with genotype 1 present opportunities for improvement in the management of this disease.

First identified by molecular cloning in 1989 (Choo, Q-L et al (1989) Science 244:359-362), HCV is now widely accepted as the most common causative agent of post-transfusion non A, non-B hepatitis (NANBH) (Kuo, G et al (1989) Science 244:362-364). Due to its genome structure and sequence homology, this virus was assigned as a new genus in the Flaviviridae family. Like the other members of the Flaviviridae, such as flaviviruses (e.g. yellow fever virus and Dengue virus types 1-4) and pestiviruses (e.g. bovine viral diarrhea virus, border disease virus, and classic swine fever virus) (Choo, Q-L et al (1989) Science 244:359-362; Miller, R.H. and R.H. Purcell (1990) Proc. Natl. Acad. Sci. USA 87:2057-2061 ), HCV is an enveloped virus containing a single strand RNA molecule of positive polarity. The HCV genome is approximately 9.6 kilobases (kb) with a long, highly conserved, noncapped 5' nontranslated region (NTR) of approximately 340 bases which functions as an internal ribosome entry site (IRES) (Wang CY et al 'An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region' RNA- A Publication of the RNA Society. 1 (5): 526-537, 1995 JuL). This element is followed by a region which encodes a single long open reading frame (ORF) encoding a polypeptide of -3000 amino acids comprising both the structural and nonstructural viral proteins.

Upon entry into the cytoplasm of the cell, this RNA is directly translated into a polypeptide of -3000 amino acids comprising both the structural and nonstructural viral proteins. This large polypeptide is subsequently processed into the individual structural and nonstructural proteins by a combination of host and virally-encoded proteinases (Rice, CM. (1996) in B.N. Fields, D.M.Knipe and P.M. Howley (eds) Virology 2^nd Edition, p931-960; Raven Press, N.Y.). Following the termination codon at the end of the long ORF, there is a 3' NTR which roughly consists of three regions: an - 40 base region which is poorly conserved among various genotypes, a variable length poly(U)/polypyrimidine tract, and a highly conserved 98 base element also called the "3' X-tail" (Kolykhalov, A. et al (1996) J. Virology 70:3363-3371 ; Tanaka, T. et al (1995) Biochem Biophys. Res. Commun. 215:744-749; Tanaka, T. et al (1996) J. Virology 70:3307-3312; Yamada, N. et al (1996) Virology 223:255-261 ). The 3' NTR is predicted to form a stable secondary structure which is essential for HCV growth in chimps and is believed to function in the initiation and regulation of viral RNA replication.

The NS5B protein (591 amino acids, 65 kDa) of HCV (Behrens, S. E. et al (1996) EMBO J. 15:12-22), encodes an RNA-dependent RNA polymerase (RdRp) activity and contains canonical motifs present in other RNA viral polymerases. The NS5B protein is fairly well conserved both intra-typically (-95-98% amino acid (aa) identity across 1 b isolates) and inter-typically (-85% aa identity between genotype 1 a and 1 b isolates). The essentiality of the HCV NS5B RdRp activity for the generation of infectious progeny virions has been formally proven in chimpanzees (A. A. Kolykhalov et al.. (2000) Journal of Virology, 74(4): 2046-2051 ). Thus, inhibition of NS5B RdRp activity (inhibition of RNA replication) is predicted to be useful to treat HCV infection.

Although the predominant HCV genotype worldwide is genotype 1 , this itself has two main subtypes, denoted 1a and 1 b. As seen from entries into the Los Alamos HCV database (www.hcv.lanl.gov) (Table 1 ) there are regional differences in the distribution of these subtypes: while genotype 1 a is most abundant in the United States, the majority of sequences in Europe and Japan are from genotype 1 b. Table 1

Based on the foregoing, there exists a significant need to identify synthetic or biological compounds for their ability to inhibit replication of both genotype 1 a and genotype 1 b of HCV.

PCT publication number WO2002/100851 generically discloses certain compounds, including certain 2-carboxy thiophene compounds, having HCV inhibitory activity. The data provided relates to an HCV polymerase assay utilising the 1 b genotype. The compounds disclosed have the formula (I)

wherein

X is chosen from -N(R³)M(R²) or -JN(R²)(R³);

M is chosen from -SO₂-, -SO-, -S-, -C(O)-, -C(S)-, -CH₂C(O)N(R⁴)-, -CH₂C(S)N(R¹⁵)-, -

CH(R¹⁵)-, -C(=N(R⁸))-, or a bond;

R⁴ is C_1-6alkyl;

R⁸ is chosen from H, Ci_i₂alkyl, C_2-i₂alkenyl, C_2-i₂alkynyl, C₆-i₄aryl, C₃-i₂heterocycle, C_3- i₂heteroaralkyl, C_6-i₆aralkyl;

R¹⁵ is chosen from H or C_1-6alkyl;

J is chosen from -C(W)-, -C(R⁶)-, -S-, -S(O)-, or -SO₂-;

W is chosen from O, S or NR⁷;

R⁷ is chosen from H, C_1-12alkyl, C_2-12alkenyl, C_2-12alkynyl, C₆-i₄aryl, C_3-12heterocycle, C_3- i₂heteroaralkyl, C_6-i₆aralkyl;

R⁶ is chosen from H, Ci_i₂alkyl, C₆-i₄aryl, or C_6-i₆aralkyl;

Y¹ is chosen from a bond, C_1-6alkyl, C_2-6alkenyl, C_2-6alkynyl;

Y is chosen from COOR¹⁶, COCOOR⁵, P(0)0ROR^b, S(O)OR⁵, S(O)₂OR⁵, tetrazole,

CON(R⁹)CH(R⁵)COOR⁵, CONR¹⁰R¹¹, CON(R⁹)-SO₂-R⁵, CONR⁹OH, or halogen;

R , R ,R ₅10 and R are each independently chosen from H, C_1-12alkyl, C_2-12alkenyl, C_2-

₁₂alkynyl, C_3-i₂heterocycle, C_3-i₈heteroaralkyl, C_6-i₈aralkyl; or R¹⁰ and R¹¹ are taken together with the nitrogen to form a 3 to 10 membered heterocycle;

R^a and R^b are each independently chosen from H, C_1-12alkyl, C_2-12alkenyl, C_2-12alkynyl, C_6- i₄aryl, C_3-i₂heterocycle, C_3-i₈heteroaralkyl, C₆-i₈aralkyl; or R^a and R^b are taken together with the oxygens to form a 5 to 10 membered heterocycle;

R¹⁶ is chosen from H, C_1-12alkyl, C_2-12alkenyl, C_2-12alkynyl, C_6-i₄aryl, C_3-12heterocycle, C_3- -isheteroaralkyl, Cβ-isaralkyl; provided that R¹⁶ is other than methyl or ethyl; R¹ is chosen from Ci.-i₂alkyl, C₂-i₂alkenyl, C₂-i₂alkynyl, C_6-i₄aryl, C_3-i₂heterocycle, C_{3- 18}heteroaralkyl, C_6-i₈aralkyl;

R² is chosen from Ci.-i₂alkyl, C_2-i₂alkynyl, C₆-i₄aryl, C_3-i₂heterocycle, C_3-i₈heteroaralkyl, C_6- i₈aralkyl;

R³ is chosen from H, C_1-12alkyl, C_2-i₂alkenyl, C_2-i₂alkynyl, C₆-i₄aryl, C_3-12heterocycle, C_3- iβheteroaralkyl, C_6-i₈aralkyl;

Z is chosen from H, halogen, or Ci_-6alkyl.

Surprisingly, it has now been found that compounds according to the present invention, generically disclosed in WO2002/100851 , and having a specific substitution pattern, exhibit improved properties over those compounds specifically disclosed in WO2002/100851.

SUMMARY OF THE INVENTION

The present invention involves novel 2-carboxy thiophene compounds represented hereinbelow, pharmaceutical compositions comprising such compounds and use of the compounds in treating viral infection, especially HCV infection.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a compound of Formula (I) :

wherein:

A represents hydroxy;

R^x represents phenyl (optionally substituted by one or more substituents selected from halo, methyl, ethyl, methoxy and trifluoromethyl); or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the thiophene;

R^γ represents phenyl (optionally substituted by one or more substituents selected from halo, methyl, ethyl, methoxy, trifluoromethyl, hydroxy and amino); or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the acetylene; bonded such that, when R^x represents phenyl or 6-membered heteroaryl, the R^y-Ξ- group is attached to R^x in the para-position to the thiophene; R² represents -Cs-zcycloalkyl (optionally substituted by one or more substituents selected from -Ci_-2alkyl optionally substituted with one or more fluoro groups, and -OH), or phenyl (optionally substituted by one or more substituents selected from halo, methyl, ethyl, methoxy and trifluoromethyl);

R³ represents -C_1-6alkyl (optionally substituted by one or more substituents selected from cyclopropyl, 5- and 6-membered heteroaryl and 5- and 6-membered heterocyclyl), -C_2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), -C_3-6cycloalkyl (optionally substituted by one or more substituents selected from -C_{1- 2}alkyl, fluoro and methoxy), or -(CH₂)theterocyclyl;

t represents 0 or 1 ;

or a salt thereof.

It is to be understood that the present invention covers all combinations of aspects, suitable and convenient groups described herein.

As used herein, "acetyl" refers to -C(O)CH₃.

As used herein unless otherwise specified, "alkyl" refers to an optionally substituted hydrocarbon group. The alkyl hydrocarbon group may be linear or branched, saturated or unsaturated. Examples of such groups include methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or hexyl and the like. Where the alkyl hydrocarbon group is unsaturated, it will be understood that there will be a minimum of 2 carbon atoms in the group, for example an alkenyl or alkynyl group. In one aspect, alkyl moieties are saturated. In one aspect, alkyl moieties are -C_1-4alkyl. Unless otherwise stated, optional substituents include -Ci_-6alkyl (unsubstituted), -C_{3- 7}cycloalkyl (unsubstituted), =CH(CH₂)_tH, fluoro, -CF₃, -OR^E, -SR^E, -C(O)NR^BR^C, -C(O)R⁰, - CO₂H, -CO₂R⁰, -NR^BR^C, -NR^AC(O)R°,

-NR^ACO₂R^D, -NR^AC(O)NR^FR^G, -SO₂NR^FR^G, -SO₂R⁰, nitro, cyano, oxo, aryl, heteroaryl and heterocyclyl.

As used herein, the term "alkenyl" refers to a linear or branched hydrocarbon group containing one or more carbon-carbon double bonds. In one aspect the alkenyl group has from 2 to 6 carbon atoms. Examples of such groups include ethenyl, propenyl, butenyl, pentenyl or hexenyl and the like.

As used herein, the term "alkynyl" refers to a linear or branched hydrocarbon group containing one or more carbon-carbon triple bonds. In one aspect the alkynyl group has from 2 to 6 carbon atoms. Examples of such groups include ethynyl, propynyl, butynyl, pentynyl or hexynyl and the like. As used herein unless otherwise specified, "cycloalkyl" refers to an optionally substituted, cyclic hydrocarbon group. The hydrocarbon group may be saturated or unsaturated, monocyclic or bridged bicyclic. Where the cycloalkyl group is saturated, examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl and the like. Where the cycloalkyl group is unsaturated, examples of such groups include cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl or cyclooctenyl and the like. In one aspect, the cycloalkyl group has from 5 to 7 carbon atoms. In one aspect, cycloalkyl moieties are cyclohexenyl, cyclopentenyl and cyclohexyl. Unless otherwise stated, the cycloalkyl group may be substituted by one or more optional substituents including -C_halky! (unsubstituted), -C^cycloalkyl (unsubstituted), =CH(CH₂)_tH, fluoro, -CF₃, -OR^E, -SR^E, - C(O)NR^BR^C, -C(O)R⁰, -CO₂H, -CO₂R⁰, -NR^BR^C, -NR^AC(0)R°, -NR^AC0₂R^D, -NR^AC(0)NR^FR^G, -SO₂NR^FR^G, -SO₂R⁰, nitro, cyano, oxo, phenyl and heterocyclyl.

As used herein, the term " alkoxy" refers to an -O-alkyl group wherein alkyl is as defined herein. Examples of such groups include methoxy, ethoxy, propoxy, butoxy, pentoxy or hexoxy and the like.

As used herein, "aryl" refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. "Aryl" includes carbocyclic aryl and biaryl groups, all of which may be optionally substituted. In one aspect, "aryl" moieties contain 6-10 carbon atoms. In one aspect, "aryl" moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted phenyl. In one aspect, unless otherwise stated, "aryl" substituents are selected from the group consisting of -Ci_-6alkyl, -C^cycloalkyl (unsubstituted), halo, -OR^E, -SR^E, -C(O)NR^BR^C, -C(O)R⁰, -CO₂H, - CO₂R⁰, -NR^BR^C, -NR^AC(0)R°,

-NR^AC0₂R°, -NR^AC(0)NR^FR^G, -SO₂NR^FR^G, -SO₂R⁰, nitro, cyano, heterocyclyl, -CF₃, -OCF₃ and phenyl.

As used herein, "carbonyl" refers to -C(O)-.

As used herein, "cyano" refers to -CN.

As used herein, "halogen" or "halo" refer to a fluorine, chlorine, bromine or iodine atom. References to "fluoro", "chloro", "bromo" or "iodo" should be construed accordingly.

As used herein, unless otherwise specified, "heteroaryl" refers to an optionally substituted, 5, 6, 8, 9 or 10 membered, aromatic group comprising one to four heteroatoms selected from N, O and S, with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. In one aspect, "heteroaryl" moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted (where applicable) pyridine, pyrazine, thiazole, thiophene, oxadiazole, oxazole, pyrimidine, pyridazine, benzodioxole, benzofuran, benzodioxin, indole, benzimidazole, benzofuran, indole, indazole, isoindole, benzothiophene, benzothiazole, benzoxazole, benzisoxazole, benzisothiazole, benzotriazole, furopyridine, furopyrimidine, furopyridazine, furopyrazine, furotriazine, pyrrolopyridine, pyrrolopyrimidine, pyrrolopyridazine, pyrrolopyrazine, pyrrolotriazine, thienopyridine, thienopyrimidine, thienopyridazine, thienopyrazine, thienotriazine, thiazolopyridine, thiazolopyrimidine, thiazolopyridazine, thiazolopyrazine, thiazolotriazine, oxazolopyridine, oxazolopyrimidine, oxazolopyridazine, oxazolopyrazine, oxazolotriazine, imidazopyridine, imidazopyrimidine, imidazopyridazine, imidazopyrazine, imidazotriazine, pyrazolopyridine, pyrazolopyrimidine, pyrazolopyridazine, pyrazolopyrazine, pyrazolotriazine, triazolopyridine, triazolopyrimidine, triazolopyridazine, triazolopyrazine, quinoline, naphthyridine, quinoxaline, quinazoline, isoquinoline, cinnoline, pyridopyridazine, pyridopyrimidine, pyridopyrazine, pyrazinopyrazine, pteridine, pyrazinopyridazine, pyrimidopyridazine, pyrimidopyrimidine, imidazothiazole, thiazolooxazole. All isomers of the above heteroaryls are within the scope of this invention. Each heteroaryl group may be attached at any ring carbon or may be attached through nitrogen when the nitrogen is part of a 5-membered ring. In one aspect, unless otherwise stated, "heteroaryl" substituents are selected from the group consisting of -Ci_-6alkyl, -C_3-7cycloalkyl (unsubstituted), halo, -OR^E, -SR^E, -C(O)NR^BR^C, -C(O)R⁰, -CO₂R⁰, - NR^BR^C, -NR^AC(O)R°, -NR^ACO₂R^D, -NR^AC(O)NR^FR^G, -SO₂NR^FR^G, -SO₂R⁰, oxo, nitro, cyano, heterocyclyl, -CF₃ and phenyl.

As used herein, "heterocyclic" and "heterocyclyl" refer to an optionally substituted, 5- or 6- membered, saturated or partially saturated, cyclic group containing 1 or 2 heteroatoms selected from N, optionally substituted by hydrogen, -Ci_-6alkyl, -C_3-7cycloalkyl (unsubstituted) -C(O)R⁰, -C(O)NR^BR^C, -C(O)OH, -SO₂R⁰, aryl or heteroaryl; O; and S, optionally substituted by one or two oxygen atoms. Ring carbon atoms may be optionally substituted by -Ci-βalkyl, -Cs-rcycloalkyl (unsubstituted), -0R^A, -C(O)R⁰, or -SO₂R⁰. In one aspect, unless otherwise stated, "heterocyclic" moieties are unsubstituted or monosubstituted tetrahydro-2H-pyran-4- yl, piperidinyl and tetrahydrofuran-3-yl.

As used herein, "nitro" refers to -NO₂.

As used herein, "oxo" refers to =0.

As used herein, "Et" refers to "ethyl", "iPr" refers to "isopropyl", "Me" refers to "methyl", "OBn" refers to "benzyloxy", and "Ph" refers to "phenyl".

R^A represents hydrogen, -C_1-6alkyl or -Cs-rcycloalkyl (unsubstituted).

R^B and R^c independently represent hydrogen, -C_1-6alkyl, -C_3-7cycloalkyl (unsubstituted), aryl, heterocyclyl or heteroaryl; or R^B and R^c together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group. R^D is selected from the group consisting of -Ci_-6alkyl, -Cs-rcycloalkyl (unsubstituted), aryl, heterocyclyl, heteroaryl, arylalkyl, and heteroarylalkyl.

R^E represents hydrogen, -Ci_-6alkyl, -Cs-zcycloalkyl (unsubstituted), arylalkyl, heteroarylalkyl, aryl, heterocyclyl or heteroaryl.

R^F and R^G are independently selected from the group consisting of hydrogen, -d-βalkyl, -C₃_ ₇cycloalkyl (unsubstituted), aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R^F and R^G together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated cyclic group.

As used herein, "arylalkyl" refers to one or more aryl groups attached to an alkyl radical. In one aspect, arylalkyl groups are benzyl or phenethyl.

As used herein, "heteroarylalkyl" refers to one or more heteroaryl groups attached to an alkyl radical. In one aspect, arylalkyl groups are pyridylmethyl or furanylmethyl.

As used herein, the term "compounds of the invention" means the compounds according to Formula I and the salts, solvates and esters thereof. The term "a compound of the invention" means any one of the compounds of the invention as defined above.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.

As used herein, the term "substituted" refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated.

In one aspect, R^x represents phenyl optionally substituted by one or more substituents selected from halo, methyl, methoxy and trifluoromethyl. In a further aspect, R^x represents 3-chlorophenyl, 3-methylphenyl or 3-fluorophenyl. In a further aspect, R^x represents unsubstituted phenyl.

In one aspect, R^γ represents thiazolyl or pyridinyl. In a further aspect, R^γ represents 1 ,3- thiazol-4-yl, 1 ,3-thiazol-2-yl, pyridin-2-yl or pyridin-3-yl. In a further aspect, R^γ represents 1 ,3-thiazol-4-yl.

In one aspect, R² represents -C_5-7cycloalkyl (optionally substituted by one or more substituents selected from -C_1-2alkyl optionally substituted with one or more fluoro groups, and -OH) or phenyl (optionally substituted by one or more substituents selected from halo).

In a further aspect, R² represents -C_5-7cycloalkyl (optionally substituted by one or more substituents selected from -Ci_-2alkyl optionally substituted with one or more fluoro groups) or phenyl (optionally substituted by one or more substituents selected from halo). In a further aspect, R² represents -C₆cycloalkyl (optionally substituted by one or more substituents selected from -Ci_-2alkyl optionally substituted with one or more fluoro groups), or phenyl (substituted by one or more chloro substituents). In a further aspect, R² represents - C₆cycloalkyl (optionally substituted by methyl or trifluoromethyl) or phenyl (substituted by one or more chloro substituents. In a further aspect, R² represents frans-4-methylcyclohexyl, frans-4-trifluoromethylcyclohexyl or 2,4-dichlorophenyl.

In one aspect, R³ represents -C_1-6alkyl (optionally substituted by one or more substituents selected from 5- and 6-membered heteroaryl and 5- and 6-membered heterocyclyl), -C_{2- 6}alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro) or -(CH₂)theterocyclyl wherein t represents 0 or 1. In a further aspect, R³ represents - C_2-4alkyl (optionally substituted by one or more substituents selected from methoxy and ethoxy), pyranyl or pyranylmethyl. In a further aspect, R³ represents unsubstituted -C_2-4alkyl, pyranyl or pyranylmethyl. In a further aspect, R³ represents 1-methylethyl, tetrahydro-2H- pyran-4-yl or tetrahydro-2H-pyran-4-ylmethyl.

In one aspect, R^x represents phenyl (optionally substituted by one or more substituents selected from halo, methyl, methoxy and trifluoromethyl); R^γ represents thiazolyl or pyridinyl;

R² represents -C_5-7cycloalkyl (optionally substituted by one or more substituents selected from -Ci_-2alkyl optionally substituted with one or more fluoro groups, and -OH) or phenyl

(optionally substituted by one or more substituents selected from halo); and R³ represents -

C_1-6alkyl (optionally substituted by one or more substituents selected from 5- and 6- membered heteroaryl and 5- and 6-membered heterocyclyl), -C_2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro) or -(CH₂)_theterocyclyl wherein t represents 0 or 1.

In a further aspect, R^x represents unsubstituted phenyl; R^γ represents 1 ,3-thiazol-4-yl, 1 ,3- thiazol-2-yl, pyridin-2-yl or pyridin-3-yl; R² represents frans-4-methylcyclohexyl, trans-4- trifluoromethylcyclohexyl or 2,4-dichlorophenyl; and R³ represents 1-methylethyl, tetrahydro- 2H-pyran-4-yl or tetrahydro-2H-pyran-4-ylmethyl.

In a further aspect, the present invention provides a compound chosen from the group consisting of:

3-[[(frans-4-Methylcyclohexyl)carbonyl](1-methylethyl)amino]-5-[4-(1 ,3-thiazol-4- ylethynyl)phenyl]-2-thiophenecarboxylic acid;

3-[[(frans-4-Methylcyclohexyl)carbonyl](tetrahydro-2H-pyran-4-ylmethyl)amino]-5-[4-(1 ,3- thiazol-4-ylethynyl)phenyl]-2-thiophenecarboxylic acid; 3-[[(frans-4-Methylcyclohexyl)carbonyl](1-methylethyl)amino]-5-[4-(2-pyridinylethynyl)phenyl]-

2-thiophenecarboxylic acid;

3-[[(frans-4-Methylcyclohexyl)carbonyl](1-methylethyl)amino]-5-[4-(1 ,3-thiazol-2- ylethynyl)phenyl]-2-thiophenecarboxylic acid;

3-[[(frans-4-Methylcyclohexyl)carbonyl](1-methylethyl)amino]-5-[4-(3-pyridinylethynyl)phenyl]- 2-thiophenecarboxylic acid;

3-(Tetrahydro-2/-/-pyran-4-yl{[frans-4-(trifluoromethyl)cyclohexyl]carbonyl}amino)-5-[4-(1 ,3- thiazol-4-ylethynyl)phenyl]-2-thiophenecarboxylic acid;

3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-[4-(1 ,3-thiazol-4-ylethynyl)phenyl]- 2-thiophenecarboxylic acid;

and salts thereof.

The compounds of Formula (I) may be in the form of their free base or pharmaceutically acceptable salts, pharmaceutically acceptable solvates or pharmaceutically acceptable esters thereof.

Also included in the present invention are pharmaceutically acceptable salt complexes. The present invention also covers the pharmaceutically acceptable salts of the compounds of Formula (I). As used herein, the term "pharmaceutically acceptable" used in relation to an ingredient (active ingredient such as an active ingredient, a salt thereof or an excipient) which may be included in a pharmaceutical formulation for administration to a patient, refers to that ingredient being acceptable in the sense of being compatible with any other ingredients present in the pharmaceutical formulation and not being deleterious to the recipient thereof. Suitable pharmaceutically acceptable salts of the compounds of Formula (I) include acid salts, for example sodium, potassium, calcium, magnesium and tetraalkylammonium and the like, or mono- or di- basic salts with the appropriate acid for example organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids and the like.

Therefore, according to a further aspect, the invention provides a pharmaceutically acceptable salt of a compound of Formula (I) and embodiments thereof.

The invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compounds of Formula (I).

The salts of a compound of Formula (I) may be isolated in solid form by conventional means from a solution thereof obtained as above. For example, a non-crystalline salt may be prepared by precipitation from solution, spray drying or freeze drying of solutions, evaporating a solution to a glass, or vacuum drying of oils, or solidification of melts obtained from reaction of the free base and the acid.

The salts of a compound of Formula (I) may be prepared by directly crystallising from a solvent in which the salt has limited solubility, or by triturating or otherwise crystallising a non-crystalline salt. For example, organic solvents such as acetone, acetonitrile, butanone, 1-butanol, ethanol, 1-propanol or tetrahydrofuran or mixtures of such solvents may be used. An improved yield of the salts may be obtained by the evaporation of some or all of the solvent or by crystallisation at elevated temperature followed by controlled cooling, for example in stages. Careful control of the precipitation temperature and seeding may be used to improve the reproducibility of the production process and the particle size distribution and form of the product.

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compound of Formula (I) are within the scope of the invention. Therefore, the present invention also relates to solvates of the compounds of Formula (I), for example hydrates.

Salts and solvates of compounds of Formula (I) which are suitable for use in medicine are those wherein the counterion or associated solvent is pharmaceutically acceptable. However, salts and solvates having non-pharmaceutically acceptable counterions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of the invention and their pharmaceutically acceptable salts and solvates.

It will be appreciated by those skilled in the art that certain protected derivatives of compounds of Formula (I), which may be made prior to a final deprotection stage, may not possess pharmacological activity as such, but may, in certain instances, be administered orally or parenterally and thereafter metabolised in the body to form compounds defined in the first aspect which are pharmacologically active. Such derivatives may therefore be described as "prodrugs". All protected derivatives and prodrugs of compounds defined in the first aspect are included within the scope of the invention. Examples of suitable pro-drugs for the compounds of the present invention are described in Drugs of Today, Volume 19, Number 9, 1983, pp 499 - 538 and in Topics in Chemistry, Chapter 31 , pp 306 - 316 and in "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985, Chapter 1 (the disclosures in which documents are incorporated herein by reference). It will further be appreciated by those skilled in the art, that certain moieties, known to those skilled in the art as "pro-moieties", for example as described by H. Bundgaard in "Design of Prodrugs" (the disclosure in which document is incorporated herein by reference) may be placed on appropriate functionalities when such functionalities are present within the compounds of Formula (I). Suitable prodrugs for compounds of the invention include : esters, carbonate esters, hemi-esters, phosphate esters, nitro esters, sulfate esters, sulfoxides, amides, carbamates, azo-compounds, phosphamides, glycosides, ethers, acetals and ketals. The present invention also relates to pharmaceutically acceptable esters of the compounds of Formula (I), for example carboxylic acid esters -COOR, in which R is selected from straight or branched chain alkyl, for example n-propyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen, -C_1-4alkyl or -Ci_-4alkoxy or amino); or for example - CH₂OC(O)R' or -CH₂OCO₂R' in which R' is alkyl (e.g. R' is f-butyl). Unless otherwise specified, any alkyl moiety present in such esters preferably contains 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters suitably comprises a phenyl group.

In one aspect, the invention provides a compound of Formula (I) in the form of parent compound, a salt or a solvate. In a further aspect, the invention provides a compound of Formula (I) in the form of parent compound or a salt thereof. In a further aspect, the invention provides a compound of Formula (I) in the form of parent compound.

Furthermore, some of the crystalline forms of the compounds of Formula (I) or salts and solvates thereof may exist in one or more polymorphic form, which are included in the present invention.

It will be appreciated that the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic, diastereoisomeric, and optically active forms. All of these racemic compounds, enantiomers and diastereoisomers are contemplated to be within the scope of the present invention. Racemic compounds may either be separated using preparative HPLC and a column with a chiral stationary phase or resolved to yield individual enantiomers utilising methods known to those skilled in the art. In addition, chiral intermediate compounds may be resolved and used to prepare chiral compounds of the invention.

It will further be appreciated that certain compounds of the present invention may exist in different tautomeric forms i.e. one or more tautomeric forms. All tautomers are contemplated to be within the scope of the present invention. For example, a claim to 2-hydroxyquinolinyl would also cover its tautomeric form, α-quinolinonyl.

Diastereoisomers of compounds the present invention may be obtained according to methods well known in the literature, for example by preparative HPLC or by chromatographic purifications. Racemic compounds may either be separated using preparative HPLC and a column with a chiral stationary phase or resolved to yield individual enantiomers utilising methods known to those skilled in the art. In addition, chiral intermediate compounds may be resolved and used to prepare chiral compounds of the invention.

The compounds of the present invention exhibit improved potency against the replication of HCV (1a and 1 b genotypes), and therefore have the potential to achieve greater efficacy in man. High potency in both genotypes is considered to be advantageous.

There is provided as a further aspect of the present invention a compound chosen from compounds of Formula (I) and pharmaceutically acceptable salts thereof for use in human or veterinary medical therapy, particularly in the treatment or prophylaxis of viral infection, particularly flavivirus infection, for example HCV infection.

It will be appreciated that reference herein to therapy and/or treatment includes, but is not limited to prevention, retardation, prophylaxis, therapy and cure of the disease. It will further be appreciated that references herein to treatment or prophylaxis of HCV infection include treatment or prophylaxis of HCV-associated disease such as liver fibrosis, cirrhosis and hepatocellular carcinoma.

In a further or alternative aspect there is provided a method for the treatment of a human or animal subject with viral infection, particularly HCV infection, which method comprises administering to said human or animal subject an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

According to another aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and/or prophylaxis of viral infection, particularly HCV infection.

PROCESSES

Compounds of Formula (I) in which A is hydroxy may be prepared from a compound of Formula (II)

in which A is a protected hydroxy group, for example an alkoxy, benzyloxy or silyloxy group and R^x, R^y, R², and R³ are as defined above for Formula (I). For example when A is methoxy or ethoxy, and R^x, R^y, R² and R³ are as defined above for Formula (I), by treatment with an appropriate base, for example aqueous sodium hydroxide or lithium hydroxide, optionally in a suitable solvent such as methanol, tetrahydrofuran or combinations thereof. Suitably, the temperature is in the range 25 to 100⁰C, for example 50 to 100⁰C. Alternatively, when A is methoxy or ethoxy and R^x, R^y, R² and R³ are as defined above for Formula (I), by treatment with lithium iodide in a suitable solvent such as pyridine, lutidine or collidine, suitably in the temperature range 100-170⁰C. For example when A is te/f-butoxy, and R^x, R^y, R² and R³ are as defined above for Formula (I), by treatment with an appropriate acid, for example trifluoroacetic acid. Suitably, the reaction is carried out in a solvent, for example dichloromethane. Suitably, the temperature is in the range 0 to 50⁰C, for example 15 to 30⁰C. For example when A is silyloxy, and R^x, R^y, R² and R³ are as defined above for Formula (I), by treatment with a suitable fluoride source for example tetrabutylammonium fluoride. The reaction is carried out in a suitable solvent, for example tetrahydrofuran. Suitably, the temperature is in the range 0 to 50⁰C, for example 15 to 30⁰C.

Therefore in one aspect, there is provided a process for the preparation of a compound of Formula (I) as defined above comprising deprotection of a compound of Formula (II)

in which A is an alkoxy, benzyloxy or silyloxy group, and R , R , R and R are as defined above for Formula (I).

Compounds of Formula (I) in which A is hydroxy, or (II) in which A is an alkoxy, benzyloxy or silyloxy group and R^x, R^y, R² and R³ are as defined above for Formula (I), may be prepared by reaction of a compound of Formula (III)

in which A is hydroxy or an alkoxy, benzyloxy or silyloxy group, and R² and R³ are as defined above for Formula (I) and X is a halo atom such as bromo or iodo; with a suitable boronic acid R^y-Ξ-R^x-B(OH)₂ or boronate ester R^y-Ξ-R^x-B(OR')(OR"), in which R' and R" are independently Ci_-6alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by C_1-6alkyl, such as a pinacol ester, in the presence of a palladium catalyst such as tetrakis(triphenylphosphine) palladium(O) or bis- [(diphenylphosphino)-ferrocene]palladium(ll) chloride, in the presence of a suitable base such as sodium carbonate, in a suitable solvent such as DMF, methanol or toluene, or combinations thereof, at a temperature in the range 50-100⁰C, optionally under an inert atmosphere. Compounds of Formula (I) in which A is hydroxy, or (II) in which A is an alkoxy, benzyloxy or silyloxy group and R^x, R^y, R² and R³ are as defined above for Formula (I), may be prepared by reaction of a compound of Formula (III)'

in which A is hydroxy or an alkoxy, benzyloxy or silyloxy group, and R² and R³ are as defined above for Formula (I) and X is a suitable boronic acid -B(OH)₂ or boronate ester - B(0R')(0R"), in which R' and R" are independently Ci_-6alkyl or R' and R" together with the oxygen atoms to which they are attached form a ring optionally substituted by C_1-6alkyl, such as a pinacol ester, with R^y-Ξ-R^x -Hal wherein Hal is a halogen such as bromide or iodide, in the presence of a palladium catalyst such as tetrakistriphenyl phosphine palladium(O) or bis- [(diphenylphosphino)-ferrocene]-palladium(ll) chloride, in the presence of a suitable base such as sodium carbonate, in a suitable solvent such as DMF or toluene, or combinations thereof, at a temperature in the range 50-100⁰C, optionally under an inert atmosphere.

Compounds of Formula (III) in which A is an alkoxy, benzyloxy or silyloxy group, X is a halo aattoomm aanndd RR²² aarnd R³ are as defined above for Formula (I), may be prepared from compounds of Formula (IV)

in which A is an alkoxy, benzyloxy or silyloxy, and R² and R³ are as defined above for Formula (I), by treatment with a suitable base such as lithium diisopropylamide and a halogen source such as bromine, iodine, N-bromosuccinide or N-iodosuccinimide in a suitable solvent such as tetrahydrofuran, and at a temperature in the range -78 to -20°C.

Compounds of Formula (III)' in which A is an alkoxy, benzyloxy or silyloxy group, X is a suitable boronic acid -B(OH)₂ or boronate ester -B(OR')(OR"), in which R' and R" are independently C_1-6alkyl or R' and R" together with the oxygen atoms to which they are attached form a ring optionally substituted by Ci_-6alkyl, and R² and R³ are as defined above for Formula (I) may be prepared from compounds of Formula (IV)

in which A is an alkoxy, benzyloxy or silyloxy, and R² and R³ are as defined above for Formula (I), by treatment with a suitable base such as lithium diisopropylamide and a boronate such as B(OR)₃ wherein R is a Ci_-6alkyl group, for example methyl, in a suitable solvent such as tetrahydrofuran, and at a temperature in the range -78°C to -20⁰C.

Compounds of Formula (III) in which A is hydroxy may be prepared from compounds of Formula (III) in which A is an alkoxy, benzyloxy or silyloxy group, for example by treatment with an appropriate base, acid or fluoride source as described in relation to the preparation of compounds of Formula (I) from compounds of Formula (II).

Compounds of Formula (IV) may a compound of Formula (V)

in which A is an alkoxy, benzyloxy or silyloxy group, and R³ is as defined above for Formula (I); with a suitable acylating agent, for example R²-C(0)-Y, wherein Y is a halo atom, for example chloro or bromo, and R² is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane, optionally in the presence of a suitable base, for example pyridine or triethylamine. A phosphine such as triphenylphosphine may optionally be used in place of the base.

Compounds of Formula (V) may be prepared by reaction of a compound of Formula (Vl)

in which A an alkoxy, benzyloxy or silyloxy group, by treatment with a suitable vinyl ether, or a suitable aldehyde or a suitable ketone, in the presence of a suitable acid, such as acetic acid, and a suitable reducing agent such as sodium triacetoxyborohydride, in a suitable solvent such as dichloromethane. Alternatively, compounds of Formula (V) may be prepared from compounds of Formula (Vl) in which A is an alkoxy, benzyloxy or silyloxy group, by treatment with a suitable alkylating agent R³-X' wherein R³ is as defined above for Formula (I) and X' is a halo atom such as chloro, bromo or iodo, or X' is a sulphonate ester such as methanesulfonate, in a suitable solvent, such as dimethylformamide, in the presence of a suitable base, such as triethylamine.

Compounds of Formula (V) may also be prepared by reacting a compound of Formula (VII)

in which A is an alkoxy, benzyloxy or silyloxy group and X is a halo atom such as bromo, with an amine R³NH₂ wherein R³ is as defined above for Formula (I), in the presence of a palladium catalyst such as tris(dibenzylidenacetone)dipalladium, in the presence of a reagent, such as 2,2'-bis(diphenylphosphino)-1 ,1 '-binaphthyl (BINAP), and a base, such as cesium carbonate, in a suitable solvent, such as toluene, and at a temperature in a suitable range, such as 80-120⁰C.

Compounds of Formula (IV) may also be prepared by reaction of a compound of Formula

in which A is an alkoxy, benzyloxy or silyloxy group, and R² is as defined above for Formula (I); with a suitable alkylating agent R³-X' in which R³ is as defined above for Formula (I) and X' is a halo atom such as chloro, bromo or iodo, or X' is a sulphonate ester such as methanesulfonate, in a suitable solvent such as dimethylformamide, in the presence of a suitable base, such as triethylamine or sodium hydride.

Compounds of Formula (VIII) may be prepared by reaction of a compound of Formula (Vl) in which A is an alkoxy, benzyloxy or silyloxy group, with a suitable acylating agent, for example R²-C(O)-Y, wherein Y is a halo atom, such as chloro or bromo, and R² is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example pyridine or triethylamine. A phosphine such as triphenylphosphine may optionally be used in place of the base.

Compounds of Formula (II) may also be prepared by reaction of a compound of Formula (IX)

in which A is an alkoxy, benzyloxy or silyloxy group, and R^x, R^y and R³ are as defined above for Formula (I), with a suitable acylating agent, for example R²-C(0)-Y, wherein Y is a halo atom, such as chloro or bromo, and R² is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane, optionally in the presence of a suitable base, for example pyridine or triethylamine. A phosphine such as triphenylphosphine may optionally be used in place of the base.

Compounds of Formula (IX) may be prepared by reaction of a compound of Formula (X)

in which X is a halo atom such as bromo or iodo and R³ is as defined above for Formula (I), with a suitable boronic acid R^y-Ξ-R^x-B(OH)₂ or boronate ester R^y-Ξ-R^x-B(OR')(OR"), in which

R^x and R^γ are as defined above for Formula (I) and R' and R" are independently C_1-6alkyl or

R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, in the presence of a palladium catalyst such as tetrakis(triphenylphosphine) palladium(O) or bis-[(diphenylphosphino)-ferrocene]palladium(ll) chloride, in the presence of a suitable base such as sodium carbonate, in a suitable solvent such as DMF, methanol or toluene, or combinations thereof, at a temperature in the range

50-100⁰C, optionally under an inert atmosphere.

Compounds of Formula (X) may be prepared by reaction of a compound of Formula (Xl)

in which A is an alkoxy, benzyloxy or silyloxy group, and X is a halo atom, such as bromo or iodo, by treatment with a suitable vinyl ether, or a suitable aldehyde or a suitable ketone, in the presence of a suitable acid, such as acetic acid, and a suitable reducing agent, such as sodium triacetoxyborohydride, in a suitable solvent, such as dichloromethane. Alternatively, compounds of Formula (X) may be prepared from compounds of Formula (Xl) in which A is an alkoxy, benzyloxy or silyloxy, and X is a halo atom, such as bromo or iodo, by treatment with a suitable alkylating agent R³-X' where R³ is as defined above for Formula (I) and X' is a halo atom such as chloro, bromo or iodo, or X' is a sulphonate ester such as methanesulfonate, in suitable solvent, such as dimethylformamide, in the presence of a suitable base, such as triethylamine.

Compounds of Formula (Xl) may be prepared by hydrolysis of a compound of Formula (XII)

in which A is an alkoxy, benzyloxy or silyloxy group and X is a halo atom, such as bromo or iodo, with a suitable base, such as aqueous potassium carbonate, optionally in the presence of an alcohol, such as methanol.

Compounds of Formula (XII) may be prepared by reaction of a compound of Formula (XIII)

in which A is an alkoxy, benzyloxy or silyloxy group, with a suitable base, such as lithium diisopropylamide and a halogen source, such as bromine, iodine, N-bromosuccinide, or N- iodosuccinimide, in a suitable solvent, such as tetrahydrofuran, at a temperature in the range -78 to -20⁰C.

Compounds of Formula (XIII) may be prepared by treating compounds of Formula (Vl) with trifluoroacetic anhydride in a suitable solvent, such as ether.

Compounds of Formula (III) in which A is an alkoxy, benzyloxy or silyloxy group may also be prepared from compounds of

in which A is an alkoxy, benzyloxy or silyloxy group, X is a halo atom, such as bromo or iodo, and R² is as defined above for Formula (I), by treatment with a suitable alkylating agent R³-X' where R³ is as defined above for Formula (I) and X' is a halo atom such as chloro, bromo or iodo, or X' is a sulphonate ester such as methanesulfonate, in a suitable solvent, such as dimethylformamide, in the presence of a suitable base, such as triethylamine or sodium hydride.

Compounds of Formula (XIV) may be prepared from compounds of Formula (Xl) by reaction with a suitable acylating agent, for example R²-C(0)-Y, wherein Y is a halo atom, such as chloro or bromo, and R² is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example pyridine or triethylamine. A phosphine such as triphenylphosphine may optionally be used in place of the base.

Compounds of Formula (Vl) and (VII) are commercially available or well known in the art.

Compounds of Formula (I) in which A is hydroxy, or (II) in which A is an alkoxy, benzyloxy or silyloxy group, may be prepared by reaction of a compound of Formula (II)'

in which Z represents a halo atom, such as chloro, bromo or iodo, and R^x, R² and R³ are as defined above for Formula (I), and A is hydroxy or an alkoxy, benzyloxy or silyloxy group, by reaction with a suitable alkyne, R^y-Ξ-H, in which R^γ is as defined above for Formula (I), in the presence of a palladium catalyst such as tetrakis(triphenylphosphine)palladium(0), a reagent such as a copper(l) halide, for example copper(l) iodide and a base such as an alkylamine, for example triethylamine or diethylamine, in a suitable solvent, such as dioxane or DMF. Alternatively, coupling methods well known in the art may be employed, see, for example, Sonogashira, K. in Metal-Catalyzed Cross-Coupling Reactions; Diederich, F., Stang, P. J., Eds.; Wiley-VCH: Weinheim, Germany, 1998; Chapter 5, pp 203-229.

Compounds of Formula (I) in which A is hydroxy, or (II) in which A is an alkoxy, benzyloxy or silyloxy group, may also be prepared by reaction of a compound of Formula (II)"

in which R^x, R² and R³ are as defined above for Formula (I), and A is hydroxy or an alkoxy, benzyloxy or silyloxy group, by reaction with a suitable heterocyclyl halide R^γ-hal, in which R^γ is as defined above for Formula (I) and suitably hal is bromo or iodo, in the presence of a palladium catalyst such as tetrakis(triphenylphosphine)palladium(0), a reagent such as a copper(l) halide, for example copper(l) iodide, and a base such as an alkylamine, for example triethylamine or diethylamine, in a suitable solvent such as dioxane or DMF. Alternatively, coupling methods well known in the art may be employed, see, for example, Sonogashira, K. in Metal-Catalyzed Cross-Coupling Reactions; Diederich, F., Stang, P. J., Eds.; Wiley-VCH: Weinheim, Germany, 1998; Chapter 5, pp 203-229.

Compounds of Formula (II)', in which Z is halo and R² and R³ are as defined above for Formula (I), may be prepared by reaction of a compound of Formula (III) with a boronic acid of Formula Z-R^x-boronic acid under the conditions described above for the preparation of compounds of Formula (I) and (II) from (III) and R¹-boronic acid.

Compounds of Formula (II)' in which Z is B(OH)₂ may be prepared by reaction of a compound of Formula (III) with a compound of Formula Z-R^X-B(OH)₂ wherein Rx is as described above for Formula (I), under the conditions described above for the preparation of compounds of Formula (I) and (II) from (III) and R¹-boronic acid.

Boronic acids Z-R^x-boronic acid, R^y-Ξ-R^x-B(OH)₂ and R^γ-boronic acid are commercially available or may be prepared by analogy to methods provided in Organometallics (1983) 2, 1316, Chem Revs. (1995) 95, 2457, Journal of Org Chem (2004) 69, 1999, SynLett (2004) (5), 892, Bioorg Med Chem (2005) 13, 2305, Tetrahedron Letters (2004) 44, 9359 and Tetrahedron Letters (2005) 45, 6657.

Boronic acid R^y-Ξ-R^x-B(OH)₂ or boronate esters R^y-Ξ-R^x-B(OR')(OR"), in which R^x and R^y are as defined above for Formula (I) and R' and R" are independently

or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by Ci_-6alkyl, such as a pinacol ester, may be prepared by reaction of an alkynyl compound R^γ-Ξ-H with a suitable halogenated boronic acid or ester, Hal-R^x-B(OH)₂ or Hal-R^x- B(OR')(OR") in the presence of potassium acetate and 1 ,1- bis(triphenylphosphino)dichloropalladium (II). The reaction may be carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example pyridine or triethylamine.

Alkynyl compounds R^γ-Ξ-H may be prepared from compounds of formula R^γ-Ξ-Si(CH₃)₃ by reaction with a suitable base, such as potassium hydroxide.

Compounds of Formula R^γ-Ξ-Si(CH₃)₃ may be prepared by coupling compounds of formula R^γ-Hal with compounds of Formula H-Ξ-Si(CH₃)₃ in the presence of a suitable catalyst such as copper (I) iodide and tetrakis(triphenylphosphine) palladium(O) or bis- [(diphenylphosphino)-ferrocene]palladium(ll) chloride.

Compounds of Formula Hal-R^x-B(OR')(OR") may be prepared from compounds of Formula 4-Hal-R^x-boronic acid by reaction with a suitable alcohol or diol in the presence of a suitable catalyst, for example an acid catalyst such as toluenesulphonic acid monohydrate. The reaction may be carried out in a suitable solvent, for example toluene. Compounds of Formula R^y-Ξ-R^x-B(OH)₂ or boronate ester R^y-Ξ-R^x-B(OR')(OR"), for use in the preparation of compounds of Formula (II) are available commercially or may be prepared from compounds of Formula R^γ-Ξ-R^x-hal or R^γ-hal by methods well known in the art.

Compounds of Formula R^γ-Ξ-R^x-hal or R^γ-hal for use in the preparation of compounds of Formula (II) are available commercially or may be prepared by methods well known in the art.

Esters of compounds of Formula (I), in which A is -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, may also be prepared by esterification of a compound of Formula (I) in which A is hydroxy by standard literature procedures for esterification.

The various general methods described above may be useful for the introduction of the desired groups at any stage in the stepwise formation of the required compound, and it will be appreciated that these general methods can be combined in different ways in such multistage processes. The sequence of the reactions in multi-stage processes should of course be chosen so that the reaction conditions used do not affect groups in the molecule which are desired in the final product.

It will be appreciated that compounds of Formula (I), (II), (II)', (II)", (III), (IV), (V), (VIII), (IX), (X) and (XIV) which exist as diastereoisomers may optionally be separated by techniques well known in the art, for example by column chromatography or recrystallisation. For example, the formation of an ester using a chiral alcohol, separation of the resulting diastereoisomers, and subsequent hydrolysis of the ester to yield the individual enantiomeric acid of Formula (I), (II), (II)', (II)", (III), (IV), (V), (VIII), (IX), (X) and (XIV).

It will be appreciated that racemic compounds of Formula (I), (II), (II)', (II)", (III), (IV), (V), (VIII), (IX), (X) and (XIV) may be optionally resolved into their individual enantiomers. Such resolutions may conveniently be accomplished by standard methods known in the art. For example, a racemic compound of Formula (I), (II), (II)', (II)", (III), (IV), (V), (VIII), (IX), (X) and (XIV) may be resolved by chiral preparative HPLC. Alternatively, racemic compounds of Formula (I), (II), (II)', (II)", (III), (IV), (V), (VIII), (IX), (X) and (XIV) which contain an appropriate acidic or basic group, such as a carboxylic acid group or amine group may be resolved by standard diastereoisomeric salt formation with a chiral base or acid reagent respectively as appropriate. Such techniques are well established in the art. For example, a racemic basic compound may be resolved by treatment with a chiral acid such as (R)-(-)- 1 ,1 '-binaphthyl-2,2'-diyl-hydrogen phosphate or (-)-di-O,O'-p-tolyl-L-tartaric acid, in a suitable solvent, for example isopropanol. The free enantiomer may then be obtained by treating the salt with a suitable base, for example triethylamine, in a suitable solvent, for example methyl te/f-butyl ether. Alternatively, racemic acid compounds may be resolved using a chiral base, for example (S)-alpha methylbenzylamine, (S)-alpha phenylethylamine, (1 S, 2S)-(+)-2- amino-1-phenyl-1 ,3-propane-diol, (-) ephidrine, quinine, brucine. Individual enantiomers of Formula (II), (II)', (II)", (III), (IV), (V), (VIII), (IX), (X) and (XIV) may then be progressed to an enantiomeric compound of Formula (I) by the chemistry described above in respect of racemic compounds.

With appropriate manipulation and protection of any chemical functionality, synthesis of compounds of Formula (I) is accomplished by methods analogous to those above and to those described in the Experimental section. Suitable protecting groups can be found, but are not restricted to, those found in T W Greene and P G M Wuts 'Protective Groups in Organic Synthesis', 3^rd Ed (1999), J Wiley and Sons.

Several of the synthetic procedures described above in general terms (and below in specific terms) may involve heating the reactants. It will be appreciated that heating may be carried out by various conventional methods but also with the use of a microwave reactor.

EXAMPLES

ABBREVIATIONS

AcOH acetic acid Biotage Biotage flash equipment for use with pre-packed silica cartridges

DCE 1 ,2-dichloroethane

DCM dichloromethane

DEF Λ/,Λ/-diethylformamide

DMF Λ/,Λ/-dimethylformamide

DME 1 ,2-dimethoxyethane

EtOAc ethyl acetate h hours

HCI hydrochloric acid

HPLC high pressure liquid chromatography

ISCO Companion Automated flash chromatography equipment with fraction analysis by UV absorption available from Presearch.

LDA lithium diisopropylamide MeCN acetonitrile MeOH methanol Mins minutes MDAP HPLC reverse phase HPLC on a Cis column using a two-solvent gradient elution with (A) water containing formic acid (0.1 %) and (B) acetonitrile-water (95:5 v/v) containing formic acid

(0.05%) as the eluents, and analysis of the fractions by electrospray mass spectroscopy.

NH2 SPE aminopropyl capped silica ion-exchange solid phase extraction cartridge

OASIS HLB cartridge Sample extraction cartridge available from Waters

SPE silica packed solid phase extraction column

TFA trifluoroacetic acid THF tetrahydrofuran

All mass spectroscopy was performed using electrospray as the method of ionisation.

Intermediate 1 Methyl 3-[(1-methylethyl)amino]-2-thiophenecarboxylate

2-Methoxypropene (9.18 ml.) was added to a solution of methyl-3-amino-2 thiophene carboxylate (5 g) in dry DCM (100 ml.) at room temperature under nitrogen. Glacial acetic acid (5.6 ml.) was added slowly. Sodium triacetoxyborohydride (10.12g) was then added in portions over 30 min. The resulting opaque solution was then left to stir at room temperature for 24 h. The mixture was poured into 8% sodium bicarbonate solution (300 ml_), the layers were separated and the DCM layer washed further with bicarbonate solution (2 x 100 ml_), dried (hydrophobic frit) and evaporated to give the title compound. MS calcd for (C₉H₁₃NO₂S+ H)⁺: 200 MS found (electrospray): (M+H)⁺ =200

Intermediate 2

Methyl 3-[[(frans-4-methylcyclohexyl)carbonyl](1-methylethyl)amino]-2- thiophenecarboxylate

Triphenylphosphine (13.9 g) was added in portions to a solution of methyl 3-[(1- methylethyl)amino]-2-thiophenecarboxylate (6.18 g) in DCM (dry, 30 ml.) at room temperature under nitrogen. frans-4-Methylcyclohexanecarbonyl chloride¹ (8.26 ml.) was added in 1 ml. portions. The solution was heated to 45⁰C under nitrogen for 2 days. A further 2 ml. of frans-4-methylcyclohexanecarbonyl chloride¹ was added and heating continued for 24 h. The mixture was cooled, poured into saturated sodium bicarbonate solution and stirred at room temperature to neutralise for 1.5 h. The layers were separated and the aqueous layer was extracted further with DCM (2 x 100 ml_). The combined organic layers were washed with saturated bicarbonate solution (2 x 100 ml_), dried (hydrophobic frit) and evaporated. Cyclohexane (100 ml.) was added and the mixture stirred for 1 h, filtered, washed with cyclohexane and air dried to give the title compound. MS calcd for (Ci₇H₂₅NO₃S+ H)⁺: 324 MS found (electrospray): (M+H)⁺ =324

Further product was obtained by purification of the filtrate. After evaporation, the residue was applied in the minimum volume of DCM to a 50 g Si SPE cartridge. Elution was with cyclohexane then cyclohexane/ethyl acetate (gradient 9:1 to 8:2). Further purification by 10 g NH2 SPE cartridge, eluting with DCM (5 x column volumes), then MeOH (5 x column volumes). The DCM fractions were combined and evaporated to give the title compound. Ref 1 : WO 2004/052885.

Alternative preparation of Intermediate 2

Method B

Methyl 3-[[(frans-4-methylcyclohexyl)carbonyl](1-methylethyl)amino]-2- thiophenecarboxylate

To a solution of methyl 3-[(1-methylethyl)amino]-2-thiophenecarboxylate (6.45 g, a synthesis of which is described as Intermediate 1 ) in DCE (100 ml.) was added Intermediate 11 (5.9 g) and the reaction was heated at 85⁰C overnight. A further portion of Intermediate 1 1 (3 g) was added and the reaction was heated at 85⁰C for 24 h. The reaction was quenched with saturated sodium bicarbonate solution. The organics were separated and the aqueous layer was extracted with DCM (x 3). The combined organics were dried by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was triturated with heptane, filtered and dried under vacuum to give the title compound. MS calcd for (C₁₇H₂₅NO₃S + H)⁺: 324 MS found (electrospray): (M+H)⁺ = 324

Intermediate 3

Methyl 5-iodo-3-[[(frans-4-methylcyclohexyl)carbonyl](1-methylethyl)amino]-2- thiophenecarboxylate

A solution of Intermediate 2 [methyl 3-[[(frans-4-methylcyclohexyl)carbonyl](1- methylethyl)amino]-2-thiophenecarboxylate] (10.5 g) in dry THF (100 ml.) was added dropwise at -77°C under nitrogen to a 2M solution of LDA in THF/heptane/ethyl benzene (48.4 ml.) maintaining an internal temperature <-70°C. The dropping funnel was washed through with dry THF (10 ml.) and stirring continued at -77°C for 2.5 hours. A solution of iodine (16.5 g) in dry THF (100 ml.) was added dropwise to the stirred reaction mixture maintaining an internal temperature <-70°C, then the dropping funnel was washed through with dry THF (10 m). After stirring under nitrogen at -77°C for 1.5 hours, the reaction mixture was quenched by addition of saturated ammonium chloride solution and warmed to 0⁰C. The mixture was diluted with 5% sodium thiosulfate solution, then the organic phase was separated and the aqueous phase was extracted with EtOAc. The combined organic phases were dried (Na₂SO₄), filtered and evaporated. The crude product was purified by flash chromatography over silica gel (Biotage) eluting with cyclohexane / EtOAc (10:1 ) to give the title compound.

MS calcd for (C₁₇H₂₄NIO₃S + H)⁺: 450 MS found (electrospray): (M+H)⁺ = 450

Intermediate 4

4-[(Trimethylsilyl)ethynyl]-1 ,3-thiazole

4-Bromo-1 ,3-thiazole (1 g), copper (I) iodide (50 mg), Pd(PPh₃)₂CI₂ (66 mg), trimethylsilyl acetylene (1.04 ml_), and triethylamine (4 ml.) were degassed and placed under a nitrogen atmosphere in a Reactivial™ , then heated at 75°C for 5 h. The reaction mixture was allowed to cool, then partitioned between DCM and water. The organic phase was dried by using a hydrophobic frit, and the solvent removed under vacuum to give an oil. This was then purified by ISCO companion silica chromatography eluting with a gradient of EtOAc /cyclohexane (0% to 25%) to give the title compound MS calcd for (C₈H₁₁ NSiS+ H)⁺: 182

MS found (electrospray): (M+H)⁺ = 182

Intermediate 5 4-Ethynyl-1 ,3-thiazole

Intermediate 4 (1.6 g) was treated with potassium hydroxide (4.2 ml_, 1 N) in methanol (13 ml.) with stirring for 3 h. The reaction mixture was diluted with water (100 ml.) and extracted with ether. The extracts were washed with brine and dried over magnesium sulphate, filtered and the solvent removed under vacuum to give an oil. This was purified by ISCO companion silica chromatography eluted with DCM to give the title compound. ¹H NMR (CDCI3): δ 8.78 (1 H d), 7.59 (1 H, d), 3.16 (1 H, s).

Intermediate 6 2-(4-lodophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

A mixture of (4-iodophenyl)boronic acid (2.4 g), 2,3-dimethyl-2,3-butanediol (6.6 g), toluenesulphonic acid monohydrate (110 mg) and in dry toluene (75 ml.) was heated under reflux for 3 h in a Dean Stark apparatus. The reaction mixture was cooled to room temperature, washed with water (100 ml_), brine, and then dried over magnesium sulphate and filtered. The solvent was evaporated to give the title compound. 1H NMR (CDCI3): δ 7.73 (2H, d), 7.52 (2H, d), 1.34 (12H, s).

Intermediate 7 4-{[4-(4,4,5,5-Tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl]ethynyl}-1 ,3-thiazole

A mixture of Intermediate 5 (709 mg), Intermediate 6 (925 mg), potassium acetate (935 mg), copper(l) iodide (30 mg) and 1 ,1-bis(triphenylphosphino) dichloropalladium (II) (95 mg) in DCM (25 ml.) was stirred and treated with triethylamine (6 ml.) for 3 h. The reaction mixture was washed with water (50 ml_), the DCM layer was separated using a hydrophobic frit and concentrated. The residue was then purified by ISCO Companion chromatography silica chromatography, eluting with a gradient of EtOAc / cyclohexane (10% to 100%) to give the title compound. 1H NMR (CDCI₃): δ 8.83 (1 H, d), 7.80 (2H, d), 7.60 (1 H, d), 7.58 (2h, d), 1.36 (12H, s).

Intermediate 8

Methyl 3-[[(frans-4-methylcyclohexyl)carbonyl](1 -methylethyl)amino]-5-[4-(1 ,3-thiazol- 4-ylethynyl)phenyl]-2-thiophenecarboxylate

Intermediate 3 (511 mg), Intermediate 7 (500 mg), sodium carbonate solution (4.45 ml_, 2N) and tetrakis(triphenylphosphine) palladium (180 mg) in DMF (8 ml.) were heated at 100°C under a nitrogen atmosphere for 4 h. The mixture was evaporated to dryness and the residue partitioned between water (100 ml.) and EtOAc (100 ml_). The aqueous phase was extracted further with EtOAc (100 ml_). The combined organic phases were washed with brine (50 ml_), dried over magnesium sulphate, filtered and evaporated and purified by ISCO companion silica chromatography eluting with EtOAc / cyclohexane (0 to 100%) to give the title compound.

MS calcd for (C₂₉H₃₂N₄O₃S+ H)⁺: 507 MS found (electrospray): (M+H)⁺ = 507

Intermediate 9

Methyl 5-(4-chlorophenyl)-3-[[(frans-4-methylcyclohexyl)carbonyl](1 - methylethyl)amino]-2-thiophenecarboxylate

A solution of frans-4-methylcyclohexanecarbonyl chloride¹ (310 mg) in DCM (dry, 3 ml.) was added slowly to a solution of methyl 5-(4-chlorophenyl)-3-[(1-methylethyl)amino]-2- thiophenecarboxylate (500 mg) in DCM (dry, 2 ml.) followed by triphenylphosphine (445 mg).

The solution was heated to 45⁰C under nitrogen for 36 h. On cooling, the mixture was diluted with DCM (20 ml_), washed with 8% sodium bicarbonate solution (2 x 20 ml_), water

(10 ml_), then 2N HCI ( 2 x 2OmL), dried (hydrophobic frit) and evaporated. This was purified by 50 g Si SPE (applying in the minimum volume of DCM); eluting with cyclohexane then cyclohexane /DCM (4:1 to 3:2 to 2:3 to 4:1 ) and finally DCM. Further elution with

EtOAc/DCM (1 :4 to 2:3 to 3:2) then EtOAc gave the title compound.

MS calcd for (C₂₃H₂₈CINO₃S+ H)⁺: 434 / 436

MS found (electrospray): (M+H)⁺ =434 / 436

Intermediate 10

Methyl 5-(4-cyanophenyl)-3-[[(frans-4-methylcyclohexyl)carbonyl](1- methylethyl)amino]-2-thiophenecarboxylate

A mixture of (4-cyanophenyl)boronic acid (59 mg), Intermediate 3 (150 mg), 2N sodium carbonate solution (0.7 ml.) and tetrakis(triphenylphosphine)palladium (0) (39 mg) in DMF (3 ml.) was heated at 100⁰C under nitrogen atmosphere, in a Reactivial™ for 90 minutes. The DMF was evaporated under vacuum and the residue partitioned between DCM and water. The DCM layer was separated using a hydrophobic frit and concentrated. The residue was purified by ISCO Companion silica chromatography, eluting with a gradient of EtOAc in cyclohexane (0% to 30%) to give the title compound. MS calcd for (C₂₄H₂₈N₂O₃S+ H)⁺: 425 MS found (electrospray): (M+H)⁺ = 425

Intermediate 11 frans^-Methylcyclohexanecarbonyl chloride

To a solution of frans-4-methylcyclohexanecarboxylic acid (40 g) in DCM (500 ml.) was added DEF (1 drop), followed by oxalyl chloride (24.5 ml.) dropwise over 1 h. The reaction mixture was stirred overnight and was evaporated in vacuo to give the title compound.

¹H NMR (CDCI₃) δ 2.65 (1 H, tt), 2.21-2.12 (2H, m), 1.86-1.78 (2H, m), 1.57-1.31 (3H, m),

1.03-0.89 (5H, m).

Intermediate 12

Methyl 3-[(tetrahydro-2H-pyran-4-ylmethyl)amino]-2-thiophenecarboxylate

To a stirred solution of methyl 3-amino-2-thiophenecarboxylate (6.28 g) in DCM (100 ml.) was added glacial acetic acid (7.2 ml.) and tetrahydro-2H-pyran-4-carbaldehyde (5 g). Sodium triacetoxyborohydride (10.2 g) was added portion-wise and the reaction mixture was stirred at room temperature overnight. A further portion of sodium triacetoxyborohydride (1 g) was added and the reaction was stirred at room temperature for 1 h. Water (100 ml.) was added and the reaction mixture was neutralised with solid sodium bicarbonate. The DCM layer was separated and the aqueous was extracted with DCM (x 2). The combined organics were dried by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-50% EtOAc in cyclohexane to give the title compound. MS calcd for (C₁₂H₁₇NO₃S + H)⁺: 256 MS found (electrospray): (M+H)⁺ = 256

Intermediate 13

Methyl S-IKfrans^-methylcyclohexylJcarbonyl^tetrahydro^H-pyran^- ylmethyl)amino]-2-thiophenecarboxylate

A mixture of Intermediate 12 (2 g) and Intermediate 11 (1.5 g) were dissolved in DCE (35 ml.) and were heated at 9O⁰C overnight. The reaction mixture was cooled and quenched with saturated sodium bicarbonate solution. The organic layer was separated and the aqueous was extracted with DCM (x 2). The combined organics were dried by passing through a hydrophobic frit and were evaporated in vacuo to give the title compound. MS calcd for (C₂₀H₂₉NO₄S + H)⁺: 380 MS found (electrospray): (M+H)⁺ = 380 Intermediate 14 Methyl S-iodo-S-IKfrans^-methylcyclohexylJcarbonyl^tetrahydro^H-pyran^- ylmethyl)amino]-2-thiophenecarboxylate

A solution of LDA (2.0M solution in THF/heptane/ethyl benzene, 16.5 ml.) was cooled to an internal temperature of -78⁰C. Intermediate 13 (4.2 g) was dissolved in dry THF (42 ml.) and was added dropwise to the cooled LDA solution, maintaining an internal temperature between -78⁰C and -7O⁰C. After stirring at -78⁰C for 15 mins, a solution of iodine (5.6 g) in dry THF (42 mL) was added, maintaining an internal temperature between -78⁰C and -7O⁰C. The reaction was stirred at -78⁰C for 10 mins, then the reaction was quenched with dropwise addition of saturated NH₄CI solution (60 mL). The reaction was allowed to warm to room temperature before being washed with 5% sodium thiosulphate solution. The organics were separated and the aqueous layer was extracted with EtOAc (x 2). The combined organics were dried over sodium sulphate and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C₂₀H₂₈INO₄S + H)⁺: 506 MS found (electrospray): (M+H)⁺ = 506

Intermediate 15

Methyl S-IKfrans^-methylcyclohexylJcarbonyl^tetrahydro^H-pyran^- ylmethyl)amino]-5-[4-(1,3-thiazol-4-ylethynyl)phenyl]-2-thiophenecarboxylate

A mixture of Intermediate 14 (200 mg), 4-{[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]ethynyl}-1 ,3-thiazole (148 mg, a synthesis of which is described as Intermediate 7), sodium carbonate (168 mg) in water (2 ml.) and tetrakis(triphenylphosphine)palladium (0) (50 mg) in DMF (6 ml.) was heated at 100⁰C for 2.5 h and was then cooled and evaporated in vacuo. The residue was partitioned between DCM and water, the organics were separated by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 10- 100% EtOAc in cyclohexane to give the title compound. MS calcd for (C₃IH₃₄N₂O₄S₂ + H)⁺: 563 MS found (electrospray): (M+H)⁺ = 563

Intermediate 16

{4-[[(frans-4-Methylcyclohexyl)carbonyl](1-methylethyl)amino]-5- [(methyloxy)carbonyl]-2-thienyl}boronic acid

A solution of LDA (2.0M solution in THF/heptane/ethyl benzene, 18.5 ml.) was cooled to - 78⁰C. Intermediate 2 (1 g) was dissolved in dry THF (60 ml.) and was added dropwise to the cooled LDA solution, maintaining an internal temperature between -78⁰C and -7O⁰C. After stirring at -78⁰C for 1 h, trimethylborate (3.8 g) was added dropwise maintaining the temperature between -78⁰C and -7O⁰C. After 10 mins the reaction was quenched with careful addition of 2N HCI (60 mL) and was allowed to warm to room temperature. The organics were separated and the aqueous was extracted with EtOAc (x 3). The combined organics were dried over sodium sulphate and evaporated in vacuo. The crude material was triturated with 1 :1 water/ isopropyl alcohol to give the title compound. MS calcd for (C₁₇H₂₆BNO₅S + H)⁺: 368 MS found (electrospray): (M+H)⁺ = 368

Intermediate 17 2-[(4-Bromophenyl)ethynyl]pyridine

A solution of 1-bromo-4-iodobenzene (1.25 g), 2-ethynylpyridine (0.50 g), copper (I) iodide (84 mg), bis(triphenylphosphino)palladium chloride (0.31 g) and triethylamine (3 mL) in dry DCM (30 mL) was stirred at room temperature under nitrogen for 1 h. The reaction was washed with water, dried using a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient of cyclohexane in DCM, followed by DCM in EtOAc to give the title compound. 1H NMR (de-DMSO) δ 8.63 (1 H, br. s), 7.87 (1 H, dt), 7.70-7.65 (3H, m), 7.59-7.55 (2H, m), 7.47-7.42 (1 H, m).

Intermediate 18

Methyl S-IKfrans^-methylcyclohexylJcarbonylKI-methylethylJaminol-S-^-^- pyridinylethynyl)phenyl]-2-thiophenecarboxylate

A mixture of Intermediate 16 (0.10 g), Intermediate 17 (77 mg), potassium phosphate (141 mg) and tetrakis(triphenylphosphine)palladium (0) (35 mg) in 1 ,4-dioxane/ water (3:1 , 2 mL) was heated in a microwave reactor at 100⁰C for 10 mins. The reaction mixture was evaporated in vacuo and the residue was dissolved in water and extracted with DCM. The organics were dried using a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C₃₀H₃₂N₂O₃S + H)⁺: 501 MS found (electrospray): (M+H)⁺ = 501

Intermediate 19

2-[(Trimethylsilyl)ethynyl]-1 ,3-thiazole

A solution of 2-bromothiazole (0.50 g), trimethylsilyl acetylene (0.475 mL), copper (I) iodide (0.06 g) and bis(triphenylphosphino)palladium chloride (0.215 g) in triethylamine (4 mL) was heated in a Reacti-vial at 75⁰C for 2 h. The reaction was repeated with fresh reagents. The two reaction mixtures were combined and poured into water and were extracted with DCM (x 2). The organics were dried using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-50% EtOAc in cyclohexane to give the title compound. MS calcd for (C₈H₁₁NSSi + H)⁺: 182 MS found (electrospray): (M+H)⁺ = 182

Intermediate 20 2-Ethynyl-1 ,3-thiazole

A mixture of Intermediate 19 (0.58 g) and potassium carbonate (0.3 g) in ethanol (20 ml.) was stirred at room temperature for 18 h, then the reaction mixture was evaporated in vacuo. The residue was dissolved in water, extracted with DCM (x 2), dried using a hydrophobic frit and evaporated to dryness to give the title compound. 1H NMR (CDCI₃) δ 7.85 (1 H, d), 7.39 (1 H, d), 3.48 (1 H, s).

Intermediate 21 2-[(4-Bromophenyl)ethynyl]-1 ,3-thiazole

A solution of 1-bromo-4-iodobenzene (0.59 g), Intermediate 20 (0.25 g), copper (I) iodide (20 mg), bis(triphenylphosphino)palladium chloride (80 mg) and triethylamine (1 ml.) in dry DCM (20 ml.) was stirred at room temperature for 2 h. The reaction was poured into water and was extracted with DCM (x 2). The combined organics were dried using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C₁₁H₆BrNS + H)⁺: 264/266 MS found (electrospray): (M+H)⁺ = 264/266

Intermediate 22

Methyl 3-[[(frans-4-methylcyclohexyl)carbonyl](1 -methylethyl)amino]-5-[4-(1 ,3-thiazol-

2-ylethynyl)phenyl]-2-thiophenecarboxylate

A mixture of Intermediate 16 (150 mg), Intermediate 21 (98 mg), tetrakis(triphenylphosphine)palladium (0) (45 mg) and potassium phosphate (0.19 g) in 1 ,4- dioxane/ water (3:1 , 2 ml.) was heated in a microwave reactor at 100⁰C for 10 mins. The reaction mixture was poured into DCM and was washed with sodium bicarbonate solution. The organics were dried using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0- 100% EtOAc in cyclohexane to give the title compound. MS calcd for (C₂₈H₃₀N₂O₃S₂ + H)⁺: 507 MS found (electrospray): (M+H)⁺ = 507

Intermediate 23 3-[(4-Bromophenyl)ethynyl]pyridine

A solution of 1-bromo-4-iodobenzene (1.25 g), 3-ethynylpyridine (0.50 g), copper (I) iodide (84 mg), bis(triphenylphosphino)palladium chloride (310 mg) and triethylamine (3 ml.) in dry DCM (30 ml.) was stirred at room temperature under nitrogen for 1 h. The reaction was washed with water, dried using a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient of cyclohexane in DCM, followed by DCM in EtOAc to give the title compound. MS calcd for (Ci₃H₈BrN + H)⁺: 258/260 MS found (electrospray): (M+H)⁺ = 258/260

Intermediate 24

Methyl S-IKfrans^-methylcyclohexylJcarbonylKI-methylethylJaminol-S-^-fS- pyridinylethynyl)phenyl]-2-thiophenecarboxylate

A mixture of Intermediate 16 (150 mg), Intermediate 23 (96 mg), tetrakis(triphenylphosphine)palladium (0) (45 mg) and potassium phosphate (190 mg) in 1 ,4- dioxane/ water (3:1 , 2 ml.) was heated in a microwave reactor at 100⁰C for 10 mins. The reaction mixture was poured into DCM and was washed with sodium bicarbonate solution.

The mixture was dried using a hydrophobic frit and was evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0- 100% EtOAc in cyclohexane to give the title compound.

MS calcd for (C₃₀H₃₂N₂O₃S + H)⁺: 501

MS found (electrospray): (M+H)⁺ = 501

Intermediate 25 Methyl 3-(tetrahydro-2H-pyran-4-ylamino)-2-thiophenecarboxylate

To a solution of methyl 3-amino-2-thiophenecarboxylate (3 g) in dry DCM (60 mL) was added tetrahydro-4H-pyran-4-one (3.822 g). Acetic acid (4.4 mL) was added slowly followed by portion-wise addition of sodium triacetoxyborohydride (8.102 g). The mixture was stirred at room temperature under nitrogen overnight. Further portions of tetrahydro-4H-pyran-4-one (1.911 g) and sodium triacetoxyborohydride (4.051 g) were added and after 4 h the reaction was neutralised with saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM and the organics were separated by hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C₁₁H₁₅NO₃S + H)⁺: 242 MS found (electrospray): (M+H)⁺ = 242 Intermediate 26 frans^-fTrifluoromethylJcyclohexanecarbonyl chloride

Oxalyl chloride (4.59 mL) was added dropwise to a solution of trans-4- (trifluoromethyl)cyclohexanecarboxylic acid² (6.85 g) in dry DCM (100 mL) at room temperature under nitrogen. After 10 mins an effervescence was observed and the reaction was stirred at room temperature overnight. The solvent was evaporated in vacuo to give the title compound.

¹H NMR (d₆-DMSO) δ 2.38-2.19 (2H, m), 1.92 (4H, dd), 1.44-1.22 (4H, m). Ref 2: DE 39 30 119 (A1 )

Intermediate 27 Methyl 3-(tetrahydro-2H-pyran-4-yl{[frans-4-

(trifluoromethyl)cyclohexyl]carbonyl}amino)-2-thiophenecarboxylate

To a solution of Intermediate 25 (500 mg) and triphenylphosphine (1.087 g) in dry DCM (2 mL) was added frans-4-(trifluoromethyl)cyclohexanecarbonyl chloride (666 mg, a synthesis of which is described as Intermediate 26). The mixture was stirred at 5O⁰C under nitrogen overnight. A further portion of frans-4-(trifluoromethyl)cyclohexanecarbonyl chloride (50 mg, a synthesis of which is described as Intermediate 26) was added and the mixture was stirred for 3 h. The reaction was partitioned between water and DCM and the aqueous was separated and washed with DCM. The combined organics were evaporated in vacuo and the crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-50% EtOAc in cyclohexane to give the title compound. MS calcd for (Ci₉H₂₄F₃NO₄S + H)⁺: 420 MS found (electrospray): (M+H)⁺ = 420

Intermediate 28

Methyl 5-iodo-3-(tetrahydro-2H-pyran-4-yl{[frans-4-

(trifluoromethyl)cyclohexyl]carbonyl}amino)-2-thiophenecarboxylate

A solution of LDA (2.0M solution in THF/heptane/ethyl benzene, 3 ml.) was cooled to an internal temperature of -79⁰C under nitrogen. Intermediate 27 (816 mg) was dissolved in dry THF (9 ml.) and was added slowly over 10 mins to the cooled LDA solution, maintaining an internal temperature between -8O⁰C and -7O⁰C. After stirring at -73⁰C for 1.75 h, a solution of iodine (1 g) in dry THF (10 mL) was added dropwise, maintaining an internal temperature below -7O⁰C. After 45 mins, the reaction was quenched with saturated NH₄CI solution (5 mL). The reaction was allowed to warm to O⁰C before being washed with 5% sodium thiosulphate solution (25 mL). The organics were separated and evaporated in vacuo, and the residue was partitioned between DCM and water. The aqueous layer was extracted with DCM (x 2) and the combined organics were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-60% EtOAc in cyclohexane to give the title compound. MS calcd for (C₁₉H₂₃F₃INO₄S + H)⁺: 546 MS found (electrospray): (M+H)⁺ = 546

Intermediate 29 Methyl 3-(tetrahydro-2H-pyran-4-yl{[frans-4-

(trifluoromethylJcyclohexyllcarbony^aminoJ-S-^I.S-thiazol^-ylethynylJphenyl]^- thiophenecarboxylate

A mixture of Intermediate 28 (30 mg), 4-{[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]ethynyl}-1 ,3-thiazole (17.1 mg, a synthesis of which is described as Intermediate 7), sodium carbonate (17.5 mg) and tetrakis(triphenylphosphine)palladium (0) (6.4 mg) in 1 ,4-dioxane (1.5 ml.) was stirred at 100⁰C for 20 mins. The mixture was evaporated in vacuo and the residue was partitioned between water and DCM. The aqueous layer was extracted with some more DCM and the combined organics were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0- 100% EtOAc in cyclohexane to give the title compound. MS calcd for (C₃₀H₂₉F₃N₂O₄S₂ + H)⁺: 603 MS found (electrospray): (M+H)⁺ = 603

Intermediate 30

Methyl 3-[[(2,4-dichlorophenyl)ca l)amino]-2-thiophenecarboxylate

To a solution of methyl 3-[(1-methylethyl)amino]-2-thiophenecarboxylate (5 g, a synthesis of which is described as Intermediate 1 ) in pyridine (45 ml.) was added 2,4-dichlorobenzoyl chloride (5.3 ml.) under nitrogen. The mixture was heated at 7O⁰C overnight under nitrogen.

The reaction was partitioned between EtOAc and saturated sodium bicarbonate solution, and the organics were separated and washed with brine. The organics were dried over sodium sulphate and evaporated in vacuo. The crude material was purified by silica Biotage cartridge, eluting with 15% EtOAc in cyclohexane to give the title compound.

MS calcd for (Ci₆H₁₅NO₃SCI₂ + H)⁺: 372/374/376

MS found (electrospray): (M+H)⁺ = 372/374/376

Intermediate 31

Methyl 3-[[(2,4-dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-iodo-2- thiophenecarboxylate

n-Butyllithium (3.49 ml_, 1.6M solution in hexanes) was added dropwise to a solution of diisopropylamine (0.781 ml.) in THF (5 ml.) at O⁰C under nitrogen. The solution was cooled to -78⁰C and a solution of Intermediate 30 (1.036 g) in THF (5 ml.) was added dropwise, maintaining an internal temperature below -65⁰C. The reaction was left to stir for 30 mins. A solution of iodine (0.846 g) in THF (5 ml.) was added dropwise, maintaining the internal temperature below -65⁰C. The reaction was left to stir for 1.5 h. The reaction was quenched with saturated ammonium chloride (10 ml.) and was allowed to warm to room temperature. EtOAc (40 ml.) was added and the layers were separated. The organics were washed with 5% sodium thiosulphate solution (3 x 30 ml_), water (30 ml.) and 2N HCI (20 ml_), and were then dried over sodium sulphate and evaporated in vacuo. The crude material was purified by silica SPE cartridge, eluting with a gradient 0-100% DCM in cyclohexane, followed by 1- 5% MeOH in DCM to give the title compound. MS calcd for (Ci₆H₁₄CI₂INO₃S + H)⁺: 498/500/502 MS found (electrospray): (M+H)⁺ = 498/500/502

Intermediate 32

Methyl 3-[[(2,4-dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-[4-(1 ,3-thiazol-4- ylethynyl)phenyl]-2-thiop

A mixture of Intermediate 31 (75 mg), 4-{[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]ethynyl}-1 ,3-thiazole (88 mg, a synthesis of which is described as Intermediate 7), tetrakis(triphenylphosphine)palladium (0) (10 mg) and sodium carbonate (63 mg) in 1 ,4- dioxane (1.5 ml.) and water (1 ml.) was heated to 110⁰C in a microwave reactor for 20 mins. The mixture was evaporated in vacuo and the residue was partitioned between DCM and 8% sodium bicarbonate solution. The organics were separated using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C₂₇H₂₀CI₂N₂O₃S₂ + H)⁺: 555/557/559 MS found (electrospray): (M+H)⁺ = 555/557/559

Intermediate 33

Methyl 5-(4-acetylphenyl)-3-[[(frans-4-methylcyclohexyl)carbonyl](1- methylethyl)amino]-2-thiophenecarboxylate

A mixture of (4-acetylphenyl)boronic acid (66 mg), methyl 5-iodo-3-[[(frans-4- methylcyclohexyl)carbonyl](1-methylethyl)amino]-2-thiophenecarboxylate (150 mg, a synthesis of which is described as Intermediate 3), 2N sodium carbonate solution (0.7 ml.) and tetrakis(triphenylphosphine)palladium (0) (39 mg) in DMF (3 ml.) was heated at 100⁰C under nitrogen atmosphere, in a Reactivial™ for 90 minutes. The DMF was evaporated in vacuo and the residue partitioned between DCM and water. The DCM layer was separated using a hydrophobic frit and concentrated. The residue was purified by ISCO Companion silica chromatography, eluting with a gradient of EtOAc in cyclohexane (0% to 30%) to give the title compound.

MS calcd for (C₂₅H_3INO₄S+ H)⁺: 442 MS found (electrospray): (M+H)⁺ = 442

Intermediate 34 5-(4-Bromophenyl)-2-(triphenylmethyl)-2H-tetrazole

A mixture of 5-(4-bromophenyl)-1 H-tetrazole (2 g), trityl chloride (2.47 g), tetrabutyl ammonium bromide (130 mg) and 2N sodium hydroxide solution (4.79 ml.) in DCM (15 ml.) was stirred vigorously at room temperature for 3 h. The mixture was diluted with DCM (50 ml.) and water (30 ml_). The layers were separated and the aqueous extracted further with DCM (3 x 20 ml_). The combined organic fractions were dried using a hydrophobic frit and evaporated in vacuo. The crude material was purified by silica SPE cartridge, eluting with DCM to give the title compound. 1H NMR (CDCI₃) δ 8.05 (2H, d), 7.6 (2H, d), 7.4-7.1 (15H, m).

Intermediate 35 Methyl 3-[[(2,4-dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-[4-(1H-tetrazol-5- yl)phenyl]-2-thiophenecarboxylate

n-Butyl lithium (2.94 ml_, 1.6M solution in hexanes) was added dropwise to a solution of 5-(4- bromophenyl)-2-(triphenylmethyl)-2H-tetrazole (2.0 g, a synthesis of which is described as Intermediate 34) in THF (25 ml.) at -78⁰C under nitrogen. The mixture was stirred at -78⁰C under nitrogen for 45 mins. Trimethoxyborate (0.669 ml.) in THF (3 ml.) was added dropwise, maintaining the internal temp at -78⁰C. This was stirred at -78⁰C for 30 mins then allowed to warm to room temperature and stirred for a further 45 mins. Water (30 ml.) was added slowly and the mixture extracted with EtOAc (2 x 40 ml_), dried over sodium sulphate and evaporated in vacuo. The material was triturated with diethyl ether (40 ml.) and the resulting solid was filtered off. Tetrakis(triphenylphosphine)palladium (0) (23 mg) was added to a mixture of methyl 3-[[(2,4-dichlorophenyl)carbonyl](1-methylethyl)amino]-5-iodo-2- thiophenecarboxylate (200 mg, a synthesis of which is described as Intermediate 31 ) and the solid (prepared above) (185 mg) in 1 ,4-dioxane (3 ml.) and 2N sodium carbonate solution (1 ml_). The mixture was heated to 100⁰C under nitrogen for 4 h. The reaction was allowed to cool and the solvent evaporated, acidified with 2N HCI and the phases separated using a hydrophobic frit. This was evaporated and the crude material was purified by silica SPE cartridge, eluting with a gradient 0-30% EtOAc in cyclohexane, followed by a gradient EtOAc to MeCN to acetone to MeOH. These latter fractions were combined and evaporated, dissolved in MeOH and applied to a NH₂ SPE cartridge. Elution was with MeOH (5 x column volumes) then 10% acetic acid/MeOH (5 x column volumes). The acetic acid/MeOH fractions were combined and evaporated in vacuo to give the title compound. MS calcd for (C₂₃Hi₉CI₂N₅O₃S + H)⁺: 516/518/520 MS found (electrospray): (M+H)⁺ = 516/518/520

Example 1

S-IKfrans^-MethylcyclohexylJcarbonyllti-methylethylJaminol-S-^-ti .S-thiazol^- ylethynyl)phenyl]-2-thiophenecarboxylic acid

lntermediate 8 (207 mg) was dissolved in THF (1.5 ml.) and methanol (1.5 ml_). Sodium hydroxide solution (1.5 ml_, 2N) was added and the mixture stirred at room temperature for 4 h. The methanol and THF were evaporated under vacuum. The residue was dissolved in water and extracted with ether (2X 50 ml_). The aqueous was then partitioned between EtOAc / 2N HCI. The organic layer was dried over magnesium sulphate, filtered and evaporated in vacuum. This was purified by SPE silica chromatography eluted with methanol / EtOAc (10% to 50%) and dried under vacuum to give the title compound. MS calcd for (C₂₉H₃₂N₄O₃S+ H)⁺: 493 MS found (electrospray): (M+H)⁺ = 493

¹H NMR (CD₃OD): δ 9.05 (1 H, d),7.90 (1 H d), 7.75 (2H, s), 7.60 (2H, d), 7.35 (1 H s), 4.85 (1 H, m), 2.18 (1 H, m), 1.85 (1 H, m)1.75-1.48 (5H, m), 1.4-1.25 (3H, m), 1.22 (3H, d), 1.01 (3H, d), 0.76 (3H, d), 0.7-0.54 (2H, m), carboxylic acid proton not seen.

Example 2

S-I^frans^-MethylcyclohexylJcarbonylJ^etrahydro^H-pyran^-ylmethylJaminol-S-^- (1 ,3-thiazol-4-ylethynyl)phenyl]-2-thiophenecarboxylic acid

To a solution of Intermediate 15 (213 mg) in ethanol (2 ml.) and THF (2 ml.) was added 2N sodium hydroxide (1 ml_). The reaction mixture was stirred overnight at room temperature.

DCM (12 ml.) was added followed by 2N HCI (6 ml.) and this was stirred for 30 mins. The organics were separated using a hydrophobic frit and were evaporated in vacuo to give the title compound.

MS calcd for (C₃₀H₃₂N₂O₄S₂ + H)⁺: 549 MS found (electrospray): (M+H)⁺ = 549

¹H NMR (CD₃OD) δ 9.04 (1 H, d), 7.89 (1 H, d), 7.75 (2H, d), 7.60 (2H, d), 7.45 (1 H, s), 3.94-

3.84 (3H, m), 3.40-3.31 (3H, m), 2.29 (1 H, tt), 1.83-1.20 (12H, m), 0.79-0.59 (5H, m), carboxylic acid proton not seen.

Example 3

S-IKfraπs^-MethylcyclohexylJcarbonyllti-methylethylJaminol-S-^^- pyridinylethynyl)phenyl]-2-thiophenecarboxylic acid

To a solution of Intermediate 18 (68 mg) in MeOH (5 mL) was added 2M lithium hydroxide (0.5 mL) and the reaction mixture was stirred at room temperature for 3 days. The reaction was evaporated in vacuo and the residue was dissolved in water. The aqueous solution was acidified to pH 6 with 2M HCI and was extracted with EtOAc. The organics were dried using a hydrophobic frit and evaporated in vacuo. The crude material was purified by MDAP HPLC and was freeze-dried from 1 ,4-dioxane to give the title compound. MS calcd for (C₂₉H₃₀N₂O₃S + H)⁺: 487 MS found (electrospray): (M+H)⁺ = 487

¹H NMR (CD₃OD) δ 13.37 (1 H, br), 8.63 (1 H, d), 7.96-7.84 (3H, m), 7.74-7.64 (4H, m), 7.44 (1 H, ddd), 4.75 (1 H, quintet), 1.97 (1 H, tt), 1.69-1.41 (5H, m), 1.32-1.11 (5H, m), 0.90 (3H, d), 0.80-0.48 (5H, m).

Example 4

S-IKfrans^-MethylcyclohexylJcarbonyllti-methylethylJaminol-S-^-ti .S-thiazol^- ylethynyl)phenyl]-2-thiophenecarboxylic acid

A solution of Intermediate 22 (102 mg) and 2M lithium hydroxide (1 mL) in MeOH (5 mL) was stirred at room temperature for 5 days. The reaction was evaporated in vacuo and the residue was dissolved in water, acidified to pH 6 with 2M HCI and extracted with EtOAc (x 2).

The combined organics were dried using a hydrophobic frit and were evaporated in vacuo.

The crude material was purified by MDAP HPLC to give the title compound.

MS calcd for (C₂₇H₂₈N₂O₃S₂ + H)⁺: 493 MS found (electrospray): (M+H)⁺ = 493

¹H NMR (d₆-DMSO) δ 13.43 (1 H, br), 8.03-7.91 (4H, m), 7.76 (2H, d), 7.70 (1 H, s), 4.75 (1 H, quintet), 1.96 (1 H, tt), 1.67-1.17 (7H, m), 1.14 (3H, d), 0.89 (3H, d), 0.74 (3H, d), 0.72-0.48

(2H, m).

Example 5

S-IKfraπs^-MethylcyclohexylJcarbonyllti-methylethylJaminol-S-^-tS- pyridinylethynyl)phenyl]-2-thiophenecarboxylic acid

A solution of Intermediate 24 (110 mg) and 2M lithium hydroxide (1 ml.) in MeOH (5 ml.) was stirred at room temperature for 5 days. The reaction mixture was evaporated in vacuo, and the residue was dissolved in water and acidified to pH 6 with 2M HCI. The mixture was extracted with EtOAc (x 2) and the organics were dried using a hydrophobic frit and evaporated in vacuo. The crude material was purified by MDAP HPLC to give the title compound.

MS calcd for (C₂₉H₃₀N₂O₃S + H)⁺: 487 MS found (electrospray): (M+H)⁺ = 487

¹H NMR (d₆-DMS0) δ 13.40 (1 H, br), 8.80 (1 H, s), 8.61 (1 H, dd), 8.02 (1 H, dt), 7.92 (2H, d), 7.72-7.67 (3H, m), 7.49 (1 H, dd), 4.75 (1 H, quintet), 1.96 (1 H, tt), 1.67-1.40 (5H, m), 1.33- 1.17 (2H, m), 1.14 (3H, d), 0.90 (3H, d), 0.78-0.47 (5H, d).

Example 6

S-tTetrahydro^H-pyran^-y^Ifrans-^trifluoromethylJcyclohexyllcarbony^aminoJ-S-^- (1,3-thiazol-4-ylethynyl)phenyl]-2-thiophenecarboxylic acid

A mixture of Intermediate 29 (26 mg), 2M sodium hydroxide solution (0.1 ml_), THF (0.1 ml.) and MeOH (0.1 ml.) was stirred at room temperature overnight. The mixture was evaporated in vacuo and the residue was partitioned between DCM and 2M HCI. The aqueous layer was extracted with more DCM and the combined organics were evaporated in vacuo. The crude material was purified by NH₂ ion exchange chromatography and was freeze dried from 1 ,4-dioxane to give the title compound. MS calcd for (C₂₉H₂₇F₃N₂O₄S₂ + H)⁺: 589 MS found (electrospray): (M+H)⁺ = 589

¹H NMR (CD₃OD) δ 9.06 (1 H, d), 7.92 (1 H, d), 7.82 (2H, d), 7.66 (2H, d), 7.50 (1 H, s), 4.76- 4.64 (1 H, m), 3.93 (2H, dd), 3.55-3.42 (2H, m), 2.22-0.88 (14H, m), carboxylic acid proton not seen. Example 7

S-I^^-DichlorophenylJcarbonylKI -methylethylJaminol-S-I^I.S-thiazol^- ylethynyl)phenyl]-2-thioph

To a solution of Intermediate 32 (50 mg) in THF (1 mL) and MeOH (1 mL) at room temperature was added 2N lithium hydroxide solution (1 mL), and the reaction was stirred for

24 h. The reaction was evaporated in vacuo and was acidified to pH 1.0 with 2N HCI. The resulting suspension was applied to an OASIS HLB cartridge, and was eluted with water (3 x column volumes) followed by MeOH (3 x column volumes). The crude material was purified by MDAP HPLC to give the title compound.

MS calcd for (C₂₆H₁₈CI₂N₂O₃S₂ + H)⁺: 541/543/545

MS found (electrospray): (M+H)⁺ = 541/543/545

¹H NMR (d₆-DMSO) δ 13.58 (1 H, br), 9.20 (1 H, d), 8.20 (1 H, d), 7.81 (2H, d), 7.74 (1 H, s),

7.71-7.63 (2H, m), 7.49 (1 H, s), 7.37-7.30 (2H, m), 4.86 (1 H, quintet), 1.39 (3H, d), 1.04 (3H, d).

Compound A

5-(4-Chlorophenyl)-3-[[(frans-4-methylcyclohexyl)carbonyl](1-methylethyl)amino]-2- thiophenecarboxylic acid

A solution of lithium hydroxide monohydrate (178 mg) in water (6 mL) was added slowly to a solution of Intermediate 9 (307 mg) in THF (2 mL) / MeOH (12 mL) and the mixture left to stir at room temperature for 24 h. The solvents were evaporated, water (40 mL) added and washed with EtOAc (2 x 20 mL). The aqueous was acidified with 2N HCI to pH 1.0 and extracted with EtOAc (3 x 2OmL). The first EtOAc extract was partitioned with 2N HCI (10 mL) and the aqueous extracted further with EtOAc (2 x 20 mL). The organic fractions were dried (Na₂SO₄) and evaporated to give the title compound. MS calcd for (C₂₂H₂₆CINO₃S+ H)⁺: 420 MS found (electrospray): (M+H)⁺ =420

¹H NMR (DMSO): δ 7.85 (2H, d), 7.60 (1 H, s), 7.54 (2H, d), 4.74 (1 H, m), 1.9-2 (1 H, m), 1.65-1.20 (7H, m), 1.13 (3H, d), 0.89 (3H, d), 0.74 (3H, d), 0.7-0.48 (2H, m), carboxylic acid proton not seen.

Compound B

5-(4-Cyanophenyl)-3-[[(frans-4-methylcyclohexyl)carbonyl](1 -methylethyl)amino]-2- thiophenecarboxylic acid

Intermediate 10 (1 13 mg) was dissolved in THF (0.7 mL) and methanol (0.7 ml_). 2N Sodium hydroxide solution (0.7 mL) was added and the mixture stirred at room temperature for 20 h.

The methanol and THF were evaporated under vacuum, and the residue was partitioned between DCM and 2N HCI solution. The DCM layer was separated using a hydrophobic frit and concentrated. The residue was purified by ISCO Companion chromatography over silica, eluting with a gradient of 0-30% EtOAc in cyclohexane (containing 0.8 % of acetic acid) to give the title compound.

MS calcd for (C₂₃H₂₆N₂O₃S+ H)⁺: 411 MS found (electrospray): (M+H)⁺ = 41 1

Compound C

5-(4-Acetylphenyl)-3-[[(frans-4-methylcyclohexyl)carbonyl](1-methylethyl)amino]-2- thiophenecarboxylic acid

Intermediate 33 (129 mg) was dissolved in THF (0.7 mL) and methanol (0.7 mL). 2N Sodium hydroxide solution (0.7 mL) was added and the mixture stirred at room temperature for 20 h. The methanol and THF were evaporated in vacuo, and the residue was partitioned between DCM and 2N HCI solution. The DCM layer was separated using a hydrophobic frit and concentrated to give the title compound. MS calcd for (C₂₄H₂₉NO₄S+ H)⁺: 428 MS found (electrospray): (M+H)⁺ = 428

¹H NMR (DMSO-d₆) δ 8.09 (2H, d), 7.90 (2H, d), 7.52 (1 H, s), 4.86 (1 H, m, partially obscured by water peak), 2.63 (3H, s), 2.08 (1 H, tt), 1.81-1.50 (5H, m), 1.44-1.19 (5H, m), 0.99 (3H, d), 0.80-0.54 (5H, m), carboxylic acid proton not seen.

Compound D S-I^^-DichlorophenylJcarbonyllti -methylethylJaminol-S-I^IH-tetrazol-S-ylJphenyl]- 2-thiophenecarboxylic acid

2N Sodium hydroxide (1 mL) was added to a solution of Intermediate 35 (50 mg) in THF (1 mL) and MeOH (2 mL). The solution was left to stir for 24 h, then evaporated and the residue acidified to pH 1.0 with 2N HCI. The resulting suspension was applied to an OASIS HLB cartridge and eluted with water (3 x column volumes) then MeOH ( 3 x column volumes). The appropriate MeOH fractions were combined and evaporated in vacuo. The crude material was purified further by MDAP HPLC to give the title compound. MS calcd for (C₂₂Hi₇CI₂N₅O₃S + H)⁺: 502/504/506 MS found (electrospray): (M+H)⁺ = 502/504/506

¹H NMR (DMSO-d₆) δ 8.15-7.96 (dd 4H), 7.8 (s, 1 H), 7.7-7.3 (m, 3H), 4.87 (1 H, m), 1.4-0.9 (dd, 6H): the carboxylic acid and tetrazole protons are assumed to be exchanged with moisture in the solvent.

The compounds according to the invention may be formulated for administration in any convenient way, and the invention therefore also includes within its scope pharmaceutical compositions for use in therapy, comprising a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof in admixture with one or more pharmaceutically acceptable diluents or carriers.

The compounds of the present invention can be administered by different routes including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical, transdermal, or transmucosal administration. For systemic administration, oral administration is preferred.

For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets and liquid preparations such as syrups, elixirs and concentrated drops. Other conventional oral dosage forms include ovules, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed-or controlled-release applications.

The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Suitable excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

Alternatively, injection (parenteral administration) may be used, e.g., intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention are formulated in liquid solutions, preferably, in pharmaceutically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced. Typical parenteral compositions consist of a solution or suspension of a compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration, for example, may be through nasal sprays, rectal suppositories, or vaginal suppositories. Typical dermal and transdermal formulations comprise a conventional aqueous or nonaqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.

Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter. A typical suppository formulation comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes or fats or their synthetic analogs.

Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the compound of Formula (I) is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

For topical administration, the compounds of the invention can be formulated into ointments, salves, gels, or creams, as is generally known in the art.

The compounds of Formula (I) can also be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e. g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134AT"") or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e. g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e. g. sorbitan trioleate.

The amounts of various compounds to be administered can be determined by standard procedures taking into account factors such as the compound (IC₅₀) potency, (EC₅₀) efficacy, and the biological half-life (of the compound), the age, size and weight of the patient, and the disease or disorder associated with the patient. The importance of these and other factors to be considered are known to those of ordinary skill in the art.

Amounts administered also depend on the routes of administration and the degree of oral bioavailability. For example, for compounds with low oral bioavailability, relatively higher doses will have to be administered. Oral administration is a preferred method of administration of the present compounds.

Suitably the composition is in unit dosage form. For oral application, for example, a tablet, or capsule may be administered, for nasal application, a metered aerosol dose may be administered, for transdermal application, a topical formulation or patch may be administered and for transmucosal delivery, a buccal patch may be administered. In each case, dosing is such that the patient may administer a single dose.

Each dosage unit for oral administration contains suitably from 0.01 to 500 mg/Kg, for example from 0.1 to 50 mg/Kg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base. The daily dosage for parenteral, nasal, oral inhalation, transmucosal or transdermal routes contains suitably from 0.01 mg to 100 mg/Kg, of a compound of Formula(l). A topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (I). The active ingredient may be administered from 1 to 6 times per day, suitably once, sufficient to exhibit the desired activity, as is readily apparent to one skilled in the art.

Compounds of Formula (I) which are active when given orally can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil. olive oil, glycerine or water with a flavoring or coloring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils, and are incorporated in a soft gelatin capsule shell.

ASSAYS

The potential for compounds of the invention to inhibit NS5B wildtype HCV polymerase activity, genotype 1 b, may be demonstrated, for example, using the following in vitro assay:

In Vitro Detection of inhibitors of HCV RNA-dependent RNA Polymerase Activity

Incorporation of [³³P]-GMP into RNA was followed by absorption of the biotin labelled RNA polymer by streptavidin containing SPA beads. A synthetic template consisting of biotinylated 13mer-oligoG hybridised to polyrC was used as a homopolymer substrate.

Genotype 1 b Full-Length Enzyme

Reaction Conditions were 0.5 μM [³³P]-GTP (20 Ci/mMol), 1 mM Dithiothreitol, 20 mM MgCI₂, 5mM MnCI₂, 20 mM Tris-HCI, pH7.5, 1.6 μg/mL polyC/0.256 μM biotinylated oligoG13, 10% glycerol, 0.01% NP-40, 0.2 u/μL RNasin and 50 mM NaCI.

HCV RNA Polymerase (Recombinant full-length NS5B (Lohmann et al, J. Virol. 71 (11 ), 1997, 8416. 'Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity') expressed in baculovirus and purified to homogeneity) was added to 4 nM final concentration.

5x concentrated assay buffer mix was prepared using 1 M MnCI₂ (0.25 ml_), glycerol (2.5ml_), 10% NP-40 (0.025 ml.) and Water (7.225 ml_), Total 10 ml_.

2x concentrated enzyme buffer contained 1 M-Tris-HCI, pH7.5 (0.4 ml_), 5M NaCI (0.2 ml_), 1 M-MgCI₂ (0.4 ml_), glycerol (1 ml_), 10% NP-40 (10 μL), 1 M DTT (20 μL) and water (7.97 ml_), Tote/ 1O mL

Substrate Mix was prepared using 5x Concentrated assay Buffer mix (4μl_), [³³P]-GTP (10 μCi/μL, 0.02μl_), 25 μM GTP (0.4 μL), 40 u/μL RNasin (0.1 μL), 20 μg/mL polyrC/biotinylated- oligorG (1.6 μL), and Water (3.94 μL), Total 10 μL.

Enzyme Mix was prepared by adding 1 mg/ml full-length NS5B polymerase (1.5 μL) to 2.81 ml. 2x-concentrated enzyme buffer.

The Assay was set up using compound (1 μL), Substrate Mix (10 μl_), and Enzyme Mix (added last to start reaction) (10 μl_), Total 21 μl_.

The reaction was performed in a U-bottomed, white, 96-well plate. The reaction was mixed on a plate-shaker, after addition of the Enzyme, and incubated for 1 h at 22°C. After this time, the reaction was stopped by addition of 40 μl_ 1.875 mg/ml streptavidin SPA beads in 0.1 M EDTA. The beads were incubated with the reaction mixture for 1 h at 22°C after which 120 μl_ 0.1 M EDTA in PBS was added. The plate was sealed, mixed centrifuged and incorporated radioactivity determined by counting in a Trilux (Wallac) or Topcount (Packard) Scintillation Counter.

After subtraction of background levels without enzyme, any reduction in the amount of radioactivity incorporated in the presence of a compound, compared to that in the absence, was taken as a measure of the level of inhibition. Ten concentrations of compounds were tested in three- or fivefold dilutions. From the counts, percentage of inhibition at highest concentration tested or IC₅₀S for the compounds were calculated using GraFit 3, GraFit 4 or

GraFit 5 software packages or a data evaluation macro for Excel based on XLFit Software (IDBS).

The potential for compounds of the invention to inhibit HCV replication, genotype 1 a and genotype 1 b, may be demonstrated, for example, using the following cell based assay:

Replicon ELISA cell based assay

Method

100 μl_ of medium containing 10% FCS were added to each well of clear, flat-bottomed 96 well microplates, excepting wells in the top row. Test compound was diluted in assay medium to twice the final required starting concentration from a 40 mM stock solution in DMSO. 200 μl_ of the starting dilution were introduced into two wells each in the top row and doubling dilutions made down the plate by the sequential transfer of 100 μl_ aliquots with thorough mixing in the wells; the final 100 μl_ were discarded. The two bottom rows were not used for compound dilutions. Huh-7 HCV replicon cell monolayers nearing confluency were stripped from growth flasks with versene-trypsin solution and the cells were resuspended in assay medium at either 2 x 10⁵ cells/mL (sub-line 5-15; genotype 1 b; Lohmann, V., Korner, F., Koch, J-O., Herian, U., Thielmann, L. and Bartenschlager, R., Science, 1999, 285, 110- 113) or at 3 x 10⁵ cells/mL (genotype 1 a; Gu, B., Gates, AT., Isken, O., Behrens, S. E. and Sarisky, R.T., J. Virol., 2003, 77, 5352-5359). 100 μl_ of cell suspension were added to all wells and the plates incubated at 37°C for 72 hours in a 5% CO₂ atmosphere. Following incubation, the assay medium was aspirated from the plates. The cell sheets were washed by gentle immersion in phosphate buffered saline (PBS), which was then aspirated off, and fixed with acetone:methanol (1 :1 ) for 5 minutes. Following a further wash with PBS, 100 μL of ELISA diluent (PBS + 0.05% v/v Tween 20 + 2% w/v skimmed milk powder) were added to all wells and the plates incubated at 37°C for 30 minutes on an orbital platform. The diluent was removed and each well then received 50 μL of a 1/200 dilution of anti-HCV specific, murine, monoclonal antibody (either Virostat #1872 or #1877), except for wells in one of the compound-free control rows which received diluent alone to act as negative controls. The plates were incubated at 37°C for 2 hours and washed 3 times with PBS/0.05% Tween 20, then 50 μL of horseradish peroxidase conjugated, anti-mouse, rabbit polyclonal serum (Dako #P0260), diluted 1/1000, were added to all wells. The plates were incubated for a further hour, the antibody removed and the cell sheets washed 5 times with PBS/Tween and blotted dry. The assay was developed by the addition of 50 μL of ortho- phenylenediamine/peroxidase substrate in urea/citrate buffer (SigmaFast, Sigma #P-9187) to each well, and colour allowed to develop for up to 15 minutes. The reaction was stopped by the addition of 25 μL per well of 2 M sulphuric acid and the plates were read at 490 nm on a Fluostar Optima spectrophotometer. The substrate solution was removed and the plates were washed in tap water, blotted dry and the cells stained with 5 % carbol fuchsin in water for 30 minutes. The stain was discarded and the cell sheets washed, dried and examined microscopically to assess cytotoxicity. Data analysis The absorbance values from all compound-free wells that had received both primary and secondary antibodies were averaged to obtain a positive control value. The mean absorbance value from the compound-free wells that had not received the primary antibody was used to provide the negative (background) control value. The readings from the duplicate wells at each compound concentration were averaged and, after the subtraction of the mean background from all values, were expressed as a percentage of the positive control signal. The quantifiable and specific reduction of expressed protein detected by the ELISA in the presence of a drug can be used as a measure of replicon inhibition. GraFit software (Erithacus Software Ltd.) was used to plot the curve of percentage inhibition against compound concentration and derive the 50% inhibitory concentration (IC₅₀) for the compound.

Results

Activity ranges

Genotype 1a Genotype 1 b

+ >5.00 i μM + >5.00 μM

++ 1.00- ^■4.99 μM ++ 1.00- 4.99 μM

+++ 0.50- -0.99μM +++ 0.50- ^■0.99μM

++++ 0.10- -0.49μM ++++ 0.10- ^■0.49μM

+++++ 0.05- -0.099 μM +++++ 0.05- ^■0.099 μM

++++++ 0.01 - - 0.049 μM ++++++ 0.01 - ^■ 0.049 μM

+++++++ 0.005 i- 0.099 μM +++++++ 0.005 - 0.099 μM

++++++++ <0.005 μM ++++++++ <0.005 μM

Compound A corresponds to the compound disclosed as Example 570 in WO2002/100851,

5-(4-chlorophenyl)-3-[isopropyl-(4-methylcyclohexanecarbonyl)amino]-thiophene-2- carboxylic acid.

Compound B corresponds to the compound disclosed as Example 576 in WO2002/100851,

5-(4-cyanophenyl)-3-[isopropyl-(4-methylcyclohexanecarbonyl)amino]-thiophene-2- carboxylic acid.

Compound C corresponds to the compound disclosed as Example 460 in WO2002/100851, 5-(4-Acetylphenyl)-3-[[(frans-4-methylcyclohexyl)carbonyl](1-methylethyl)amino]-2- thiophenecarboxylic acid.

Compound D corresponds to the compound disclosed as Example 430 in WO2002/100851, 3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-[4-(1/-/-tetrazol-5-yl)phenyl]-2- thiophenecarboxylic acid

Compounds A, B, C and D may be made according to the processes described in WO2002/100851 or as described hereinabove.

The compounds of the present invention which have been tested demonstrate a surprisingly superior potency as HCV polymerase inhibitors, as shown by the IC₅₀ values in the cell- based assays across both of the 1a and 1 b genotypes of HCV, compared to Compounds A, B, C and D. Accordingly, the compounds of the present invention are of great potential therapeutic benefit in the treatment and prophylaxis of HCV.

When a compound of Formula (I) or a salt, solvate or ester thereof is used in combination with a second therapeutic agent active against the same disease state, the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. It will be appreciated that the amount of a compound of Formula (I) or a salt, solvate or ester thereof required for use in treatment will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian. The pharmaceutical compositions according to the invention may also be used in combination with other therapeutic agents, for example immune therapies [eg. interferon, such as Interferon alfa-2a (ROFERONO-A; Hoffmann-La Roche), inteferon alpha-2b (INTRONOA; Schering-Plough), interferon alfacon-1 (INFERGEN®; Intermune), peginterferon alpha-2b (PEGI NTRON ™; Schering-Plough) or peginterferon alpha-2a (PEGASYS®; Hoffmann-La Roche)], therapeutic vaccines, antifibrotic agents, anti-inflammatory agents (such as corticosteroids or NSAIDs), bronchodilators [such as beta-2 adrenergic agonists and xanthines (e.g. theophylline)], mucolytic agents, anti-muscarinics, anti-leukotrienes, inhibitors of cell adhesion (e.g. ICAM antagonists), anti-oxidants (eg N-acetylcysteine), cytokine agonists, cytokine antagonists, lung surfactants and/or antimicrobial, anti-viral agents (e.g. ribavirin and amantidine), and anti-HCV agents [e.g. HCV NS3 protease inhibitors, e.g. . VX950 (telapravir; Vertex) or SCH503034 (Schering Plough)], or HCV NS5b polymerase inhibitors [for example HCV796 (Wyeth) or R1626 (Roche)], RNAi agents or cyclophilin inhibitors. The compositions according to the invention may also be used in combination with gene replacement therapy.

The invention thus provides, in a further aspect, a combination comprising at least one compound of Formula (I) together with at least one other therapeutically active agent, especially interferon, ribavirin and/or an additional anti-HCV agent. The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof represent a further aspect of the invention.

The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. Appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art.

All publications, including but not limited to patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference as though fully set forth.

Claims

Claims

1. A compound of Formula (I) :

wherein:

A represents hydroxy;

R^x represents phenyl (optionally substituted by one or more substitutents selected from halo, methyl, ethyl, methoxy and trifluoromethyl); or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the thiophene;

R^γ represents phenyl (optionally substituted by one or more substitutents selected from halo, methyl, ethyl, methoxy, trifluoromethyl, hydroxy and amino); or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the acetylene; bonded such that, when R^x represents phenyl or 6-membered heteroaryl, the R^y-Ξ- group is attached to R^x in the para-position to the thiophene;

R² represents -C_5-7cycloalkyl (optionally substituted by one or more substitutents selected from -Ci_-2alkyl optionally substituted with one or more fluoro groups, and -OH), or phenyl (optionally substituted by one or more substitutents selected from halo, methyl, ethyl, methoxy and trifluoromethyl);

R³ represents linear or branched -C_1-6alkyl (optionally substituted by one or more substituents selected from cyclopropyl, 5- and 6-membered heteroaryl and 5- and 6- membered heterocyclyl), linear or branched -C_2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), -Cs-ecycloalkyl (optionally substituted by one or more substitutents selected from -d^alkyl, fluoro and methoxy), or - (CH₂)theterocyclyl;

t represents 0 or 1 ;

or a salt thereof.

2. A compound as claimed in claim 1 chosen from compounds of Formula (I) selected from the group consisting of: 3-[[(frans-4-Methylcyclohexyl)carbonyl](1-methylethyl)amino]-5-[4-(1 ,3-thiazol-4- ylethynyl)phenyl]-2-thiophenecarboxylic acid;

3-[[(frans-4-Methylcyclohexyl)carbonyl](tetrahydro-2/-/-pyran-4-ylmethyl)amino]-5-[4-(1 ,3- thiazol-4-ylethynyl)phenyl]-2-thiophenecarboxylic acid;

3-[[(frans-4-Methylcyclohexyl)carbonyl](1-methylethyl)amino]-5-[4-(2-pyridinylethynyl)phenyl]- 2-thiophenecarboxylic acid;

3-[[(frans-4-Methylcyclohexyl)carbonyl](1-methylethyl)amino]-5-[4-(1 ,3-thiazol-2- ylethynyl)phenyl]-2-thiophenecarboxylic acid;

3-[[(frans-4-Methylcyclohexyl)carbonyl](1-methylethyl)amino]-5-[4-(3-pyridinylethynyl)phenyl]-

2-thiophenecarboxylic acid; 3-(Tetrahydro-2/-/-pyran-4-yl{[frans-4-(trifluoromethyl)cyclohexyl]carbonyl}amino)-5-[4-(1 ,3- thiazol-4-ylethynyl)phenyl]-2-thiophenecarboxylic acid;

3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-[4-(1 ,3-thiazol-4-ylethynyl)phenyl]-

2-thiophenecarboxylic acid; and salts thereof.

3. A compound as claimed in Claim 1 , wherein R^x represents phenyl optionally substituted by one or more substitutents selected from halo, methyl, methoxy and trifluoromethyl.

4. A compound as claimed in Claim 1 or 3, wherein R^γ represents thiazolyl or pyridinyl.

5. A compound as claimed in Claim 1 , 3 or 4, wherein R² represents -Cs^cycloalkyl (optionally substituted by one or more substitutents selected from -Ci_-2alkyl optionally substituted with one or more fluoro groups, and -OH) or phenyl (optionally substituted by one or more substituents selected from halo).

6. A compound as claimed in Claim 1 , 3, 4 or 5, wherein R³ represents -C_1-6alkyl (optionally substituted by one or more substituents selected from 5- and 6-membered heteroaryl and 5- and 6-membered heterocyclyl), -C_2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy or fluoro) or -(CH₂)_theterocyclyl wherein t represents 0 or 1 ,

7. A method of treating or preventing viral infection which comprises administering to a subject in need thereof, an effective amount of a compound of Formula (I):

wherein A, R^x, R^γ, R² and R³ are as defined in claim 1 , or a pharmaceutically acceptable salt thereof.

8. A method as claimed in claim 7 which involves inhibiting HCV replication.

9. A compound of Formula (I):

wherein A, R^x, R^γ, R² and R³ are as defined in claim 1 , or a pharmaceutically acceptable salt thereof, for use in medical therapy.

10. A compound as claimed in claim 9 wherein the medical therapy is the treatment of viral infection.

11. A compound as claimed in claim 10 wherein the viral infection is HCV.

12. Use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof as defined in claim 1 , in the manufacture of a medicament for the treatment and/or prophylaxis of viral infection.

13. Use as claimed in claim 12 wherein the viral infection is HCV.

14. A pharmaceutical formulation comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof as defined in claim 1 in conjunction with at least one pharmaceutically acceptable diluent or carrier.

15. A combination comprising a compound of Formula (I) as defined in Claim 1 , together with at least one other therapeutically active agent.

16. A combination as claimed in Claim 15, wherein the other therapeutically active agent is selected from interferon, ribavirin and/or an additional anti-HCV agent.